Inhibitors of checkpoint kinases

ABSTRACT

The instant invention provides for compounds which comprise fused pyrazoles that inhibit CHK1 activity. The invention also provides for compositions comprising such inhibitory compounds and methods of inhibiting CHK1 activity by administering the compound to a patient in need of treatment of cancer.

PRIORITY CLAIM

This application is a §371 application of PCT/US06/049506 that was filedon Dec. 29, 2006, which claims priority from the U.S. ProvisionalApplication No. 60/755,986, filed on Jan. 4, 2006, now expired.

BACKGROUND OF THE INVENTION

Cell cycle checkpoints are regulatory pathways that control the orderand timing of cell cycle transitions. They ensure that critical eventssuch as DNA replication and chromosome segregation are completed in highfidelity. The regulation of these cell cycle checkpoints is a criticaldeterminant of the manner in which tumor cells respond to manychemotherapies and radiation. Many effective cancer therapies work bycausing DNA damage; however, resistance to these agents remains asignificant limitation in the treatment of cancer. Of the severalmechanisms of drug resistance, an important one is attributed to theprevention of cell cycle progression through the control of criticalactivation of a checkpoint pathway. This arrests the cell cycle toprovide time for repair, and induces the transcription of genes tofacilitate repair, thereby avoiding immediate cell death. By abrogatingcheckpoint arrests at, for example, the G2 checkpoint, it may bepossible to synergistically augment tumor cell death induced by DNAdamage and circumvent resistance.

Human CHK1 plays a role in regulating cell cycle arrest byphosphorylating the phosphatase cdc25 on Serine 216, which may beinvolved in preventing activation of cdc2/cyclin B and initiatingmitosis. Therefore, inhibition of CHK1 should enhance DNA damagingagents by initiating mitosis before DNA repair is complete and therebycausing tumor cell death. It is an object of the instant invention toprovide novel compounds that are inhibitors of CHK1 (also referred to asChek1).

It is also an object of the present invention to provide pharmaceuticalcompositions that comprise the novel compounds that are inhibitors ofCHK1.

It is also an object of the present invention to provide a method fortreating cancer that comprises administering such inhibitors of CHK1activity.

SUMMARY OF THE INVENTION

The instant invention provides for compounds which comprise fusedpyrazoles that inhibit CHK1 activity. The invention also provides forcompositions comprising such inhibitory compounds and methods ofinhibiting CHK1 activity by administering the compound to a patient inneed of treatment of cancer. These fused pyrazoles have improvedhemodynamic properties when compared to other fused pyrazoles reportedin U.S. Application No. 60/691,694.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the instant invention are useful in the inhibition ofthe activity of CHK1. In an embodiment of this invention, the inhibitorsof CHK1 activity are illustrated by the Formula A:

wherein:

R¹ is independently selected from H and F, wherein at least one of R¹ isF;

or a pharmaceutically acceptable salt or a stereoisomer thereof.

Specific compounds of the instant invention are:

-   5-(3-amino-2-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one    (1-12a);-   5-(3-amino-2-(R)-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one    (1-15);-   5-(3-amino-2-(S)-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one    (1-16); and-   5-(3-amino-2,2-difluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one    (1-17);    or a pharmaceutically acceptable salt or a stereoisomer thereof.

HCl salts of the instant invention are:

-   5-(3-amino-2-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one    (1-12a);-   5-(3-amino-2-(R)-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one    (1-15); and-   5-(3-amino-2-(S)-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one    (1-16);    or a stereoisomer thereof.

A TFA salt of the instant invention is:

-   5-(3-amino-2,2-difluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one    (1-17);    or a stereoisomer thereof.

The compounds of the present invention may have asymmetric centers,chiral axes, and chiral planes (as described in: E. L. Eliel and S. H.Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York,1994, pages 1119-1190), and occur as racemates, racemic mixtures, and asindividual diastereomers, with all possible isomers and mixturesthereof, including optical isomers, being included in the presentinvention. In addition, the compounds disclosed herein may exist astautomers and both tautomeric forms are intended to be encompassed bythe scope of the invention, even though only one tautomeric structure isdepicted.

It is understood that one or more silicon (Si) atoms can be incorporatedinto the compounds of the instant invention in place of one or morecarbon atoms by one of ordinary skill in the art to provide compoundsthat are chemically stable and that can be readily synthesized bytechniques known in the art from readily available starting materials.Carbon and silicon differ in their covalent radius leading todifferences in bond distance and the steric arrangement when comparinganalogous C-element and Si-element bonds. These differences lead tosubtle changes in the size and shape of silicon-containing compoundswhen compared to carbon. One of ordinary skill in the art wouldunderstand that size and shape differences can lead to subtle ordramatic changes in potency, solubility, lack of off target activity,packaging properties, and so on. (Diass, J. O. et al. Organometallics(2006) 5:1188-1198; Showell, G. A. et al. Bioorganic & MedicinalChemistry Letters (2006) 16:2555-2558).

Included in the instant invention is the free form of compounds ofFormula A, as well as the pharmaceutically acceptable salts andstereoisomers thereof. Some of the isolated specific compoundsexemplified herein are the protonated salts of amine compounds. The term“free form” refers to the amine compounds in non-salt form. Theencompassed pharmaceutically acceptable salts not only include theisolated salts exemplified for the specific compounds described herein,but also all the typical pharmaceutically acceptable salts of the freeform of compounds of Formula A. The free form of the specific saltcompounds described may be isolated using techniques known in the art.For example, the free form may be regenerated by treating the salt witha suitable dilute aqueous base solution such as dilute aqueous NaOH,potassium carbonate, ammonia and sodium bicarbonate. The free forms maydiffer from their respective salt forms somewhat in certain physicalproperties, such as solubility in polar solvents, but the acid and basesalts are otherwise pharmaceutically equivalent to their respective freeforms for purposes of the invention.

The pharmaceutically acceptable salts of the instant compounds can besynthesized from the compounds of this invention which contain a basicor acidic moiety by conventional chemical methods. Generally, the saltsof the basic compounds are prepared either by ion exchangechromatography or by reacting the free base with stoichiometric amountsor with an excess of the desired salt-forming inorganic or organic acidin a suitable solvent or various combinations of solvents. Similarly,the salts of the acidic compounds are formed by reactions with theappropriate inorganic or organic base.

Thus, pharmaceutically acceptable salts of the compounds of thisinvention include the conventional non-toxic salts of the compounds ofthis invention as formed by reacting a basic instant compound with aninorganic or organic acid. For example, conventional non-toxic saltsinclude those derived from inorganic acids such as hydrochloric,hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like, aswell as salts prepared from organic acids such as acetic, propionic,succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic,pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic,salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic,methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic:(TFA) and the like.

When the compound of the present invention is acidic, suitable“pharmaceutically acceptable salts” refers to salts prepared formpharmaceutically acceptable non-toxic bases including inorganic basesand organic bases. Salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, zinc and the like. Particularlypreferred are the ammonium, calcium, magnesium, potassium and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as arginine, betainecaffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylaminetripropylamine, tromethamine and the like.

The preparation of the pharmaceutically acceptable salts described aboveand other typical pharmaceutically acceptable salts is more fullydescribed by Berg et al, “Pharmaceutical Salts,” J. Pharm. Sci.,1977:66:1-19.

It will also be noted that the compounds of the present invention arepotentially internal salts or zwitterions, since under physiologicalconditions a deprotonated acidic moiety in the compound, such as acarboxyl group, may be anionic, and this electronic charge might then bebalanced off internally against the cationic charge of a protonated oralkylated basic moiety, such as a quaternary nitrogen atom.

Utility

The compounds, compositions and methods provided herein are particularlydeemed useful for the treatment of cancer. Cancers that may be treatedby the compounds, compositions and methods of the invention include, butare not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma,rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma andteratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiatedsmall cell, undifferentiated large cell, adenocarcinoma), alveolar(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,chondromatous hamartoma, mesothelioma, non-small cell lung;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma)colon, colorectal, rectal; Genitourinary tract: kidney (adenocarcinoma,Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra(squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma),prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma,embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma,interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors,lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma,hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone:osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological:uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma [serouscystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast;Hematologic: blood (myeloid leukemia [acute and chronic], acutelymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferativediseases, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma [malignant lymphoma]; Skin: malignantmelanoma, basal cell carcinoma, squamous cell carcinoma, Karposi'ssarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma,keloids, psoriasis; and Adrenal glands: neuroblastoma. Thus, the term“cancerous cell” as provided herein, includes a cell afflicted by anyone of the above-identified conditions.

Cancers that may be treated by the compounds, compositions and methodsof the invention include, but are not limited to: breast, prostate,colon, colorectal, lung, non-small cell lung, brain, testicular,stomach, pancrease, skin, small intestine, large intestine, throat, headand neck, oral, bone, liver, bladder, kidney, thyroid and blood.

Cancers that may be treated by the compounds, compositions and methodsof the invention include: breast, prostate, colon, ovarian, colorectaland lung.

Cancers that may be treated by the compounds, compositions and methodsof the invention include: breast, colon, (colorectal) and lung.

Cancers that may be treated by the compounds, compositions and methodsof the invention include: lymphoma and leukemia.

The compounds of the invention are also useful in preparing a medicamentthat is useful in treating cancer.

The compounds of this invention may be administered to mammals,including humans, either alone or, in combination with pharmaceuticallyacceptable carriers, excipients or diluents, in a pharmaceuticalcomposition, according to standard pharmaceutical practice. Thecompounds can be administered orally or parenterally, including theintravenous, intramuscular, intraperitoneal, subcutaneous, rectal andtopical routes of administration.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, microcrystalline cellulose, sodiumcrosscarmellose, corn starch, or alginic acid; binding agents, forexample starch, gelatin, polyvinyl-pyrrolidone or acacia, andlubricating agents, for example, magnesium stearate, stearic acid ortalc. The tablets may be uncoated or they may be coated by knowntechniques to mask the unpleasant taste of the drug or delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a watersoluble taste masking material such as hydroxypropylmethyl-cellulose orhydroxypropylcellulose, or a time delay material such as ethylcellulose, cellulose acetate buryrate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with watersoluble carrier such as polyethyleneglycol or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active material in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethylene-oxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose, saccharin or aspartame.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present. These compositions may be preserved by theaddition of an anti-oxidant such as ascorbic acid.

The pharmaceutical compositions of the invention may also be in the formof an oil-in-water emulsion. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring phosphatides, for example soy bean lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening, flavouring agents, preservatives and antioxidants.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative, flavoring and coloring agentsand antioxidant.

The pharmaceutical compositions may be in the form of sterile injectableaqueous solutions. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution and isotonic sodium chloridesolution.

The sterile injectable preparation may also be a sterile injectableoil-in-water microemulsion where the active ingredient is dissolved inthe oily phase. For example, the active ingredient may be firstdissolved in a mixture of soybean oil and lecithin. The oil solutionthen introduced into a water and glycerol mixture and processed to forma microemulation.

The injectable solutions or microemulsions may be introduced into apatient's blood-stream by local bolus injection. Alternatively, it maybe advantageous to administer the solution or microemulsion in such away as to maintain a constant circulating concentration of the instantcompound. In order to maintain such a constant concentration, acontinuous intravenous delivery device may be utilized. An example ofsuch a device is the Deltec CADD-PLUS™ model 5400 intravenous pump.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension for intramuscular andsubcutaneous administration. This suspension may be formulated accordingto the known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butane diol. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectables.

Compounds of the instant invention may also be administered in the formof suppositories for rectal administration of the drug. Thesecompositions can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter, glycerinatedgelatin, hydrogenated vegetable oils, mixtures of polyethylene glycolsof various molecular weights and fatty acid esters of polyethyleneglycol.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing the compound of Formula A are employed. (For purposesof this application, topical application shall include mouth washes andgargles.)

The compounds for the present invention can be administered inintranasal form via topical use of suitable intranasal vehicles anddelivery devices, or via transdermal routes, using those forms oftransdermal skin patches well known to those of ordinary skill in theart. To be administered in the form of a transdermal delivery system,the dosage administration will, of course, be continuous rather thanintermittent throughout the dosage regimen. Compounds of the presentinvention may also be delivered as a suppository employing bases such ascocoa butter, glycerinated gelatin, hydrogenated vegetable oils,mixtures of polyethylene glycols of various molecular weights and fattyacid esters of polyethylene glycol.

When a composition according to this invention is administered into ahuman subject, the daily dosage will normally be determined by theprescribing physician with the dosage generally varying according to theage, weight, and response of the individual patient, as well as theseverity of the patient's symptoms.

In an embodiment, a suitable amount of an inhibitor of CHK1 isadministered to a mammal undergoing treatment for cancer. Administrationoccurs in an amount of inhibitor of between about 0.1 mg/kg of bodyweight to about 60 mg/kg of body weight per day, or between 0.5 mg/kg ofbody weight to about 40 mg/kg of body weight per day. Anothertherapeutic dosage that comprises the instant composition includes fromabout 0.01 mg to about 1000 mg of inhibitor of CHK1. In anotherembodiment, the dosage comprises from about 1 mg to about 1000 mg ofinhibitor of CHK1.

The instant compounds are also useful in combination with therapeutic,chemotherapeutic and anti-cancer agents. Combinations of the presentlydisclosed compounds with therapeutic, chemotherapeutic and anti-canceragents are within the scope of the invention. Examples of such agentscan be found in Cancer Principles and Practice of Oncology by V. T.Devita and S. Hellman (editors), 6^(th) edition (Feb. 15, 2001),Lippincott Williams & Wilkins Publishers. A person of ordinary skill inthe art would be able to discern which combinations of agents would beuseful based on the particular characteristics of the drugs and thecancer involved. Such agents include the following: estrogen receptormodulators, androgen receptor modulators, retinoid receptor modulators,cytotoxic/cytostatic agents, antiproliferative agents, prenyl-proteintransferase inhibitors, HMG-CoA reductase inhibitors and otherangiogenesis inhibitors, HIV protease inhibitors, reverse transcriptaseinhibitors, inhibitors of cell proliferation and survival signaling,bisphosphonates, aromatase inhibitors, siRNA therapeutics, γ-secretaseinhibitors, agents that interfere with receptor tyrosine kinases (RTEKs)and agents that interfere with cell cycle checkpoints. The instantcompounds are particularly useful when co-administered with radiationtherapy.

“Estrogen receptor modulators” refers to compounds that interfere withor inhibit the binding of estrogen to the receptor, regardless ofmechanism. Examples of estrogen receptor modulators include, but are notlimited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081,toremifene, fulvestrant,4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

“Androgen receptor modulators” refers to compounds which interfere orinhibit the binding of androgens to the receptor, regardless ofmechanism. Examples of androgen receptor modulators include finasterideand other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide,liarozole, and abiraterone acetate.

“Retinoid receptor modulators” refers to compounds which interfere orinhibit the binding of retinoids to the receptor, regardless ofmechanism. Examples of such retinoid receptor modulators includebexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid,α-difluoromethylomithine, ILX23-7553, trans-N-(4′-hydroxyphenyl)retinamide, and N-4-carboxyphenyl retinamide.

“Cytotoxic/cytostatic agents” refer to compounds which cause cell deathor inhibit cell proliferation primarily by interfering directly with thecell's functioning or inhibit or interfere with cell myosis, includingalkylating agents, tumor necrosis factors, intercalators, hypoxiaactivatable compounds, microtubule inhibitors/microtubule-stabilizingagents, inhibitors of mitotic kinesins, histone deacetylase inhibitors,inhibitors of kinases involved in mitotic progression, inhibitors ofkinases involved in growth factor and cytokine signal transductionpathways, antimetabolites, biological response modifiers,hormonal/anti-hormonal therapeutic agents, haematopoietic growthfactors, monoclonal antibody targeted therapeutic agents, topoisomeraseinhibitors, proteosome inhibitors, ubiquitin ligase inhibitors, andaurora kinase inhibitors.

Examples of cytotoxic/cytostatic agents include, but are not limited to,sertenef, cachectin, ifosfamide, tasonermin, lonidamine, carboplatin,altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine,nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine,improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride,pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, irofulven,dexifosfamide, cis-aminedichloro(2-methyl-pyridine)platinum,benzylguanine, glufosfamide, GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum(II)]tetrachloride,diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin,pinafide, valrubicin, amrubicin, antineoplaston,3′-deamino-3′-morpholino-13-deoxo-10-hydroxycaminomycin, annamycin,galarubicin, elinafide, MEN10755,4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin (seeWO 00/50032), Raf kinase inhibitors (such as Bay43-9006) and mTORinhibitors (such as Wyeth's CCI-779).

An example of a hypoxia activatable compound is tirapazamine.

Examples of proteosome inhibitors include but are not limited tolactacystin and MLN-341 (Velcade).

Examples of microtubule inhibitors/microtubule-stabilising agentsinclude paclitaxel, vindesine sulfate,3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxol, rhizoxin,dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881,BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide,TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and6,288,237) and BMS188797. In an embodiment the epothilones are notincluded in the microtubule inhibitors/microtubule-stabilising agents.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine,irinotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)propanamine,1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione,lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350,BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane,2′-dimethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide,asulacrine,(5a,5aB,8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydroxy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium,6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one,N-[1[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide,N-(2-(dimethylamino)ethyl)acridine-4-carboxamide,6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one,and dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the humanmitotic kinesin KSP, are described in Publications WO03/039460,WO03/050064, WO03/050122, WO03/049527, WO03/049679, WO03/049678,WO04/039774, WO03/079973, WO03/099211, WO03/105855, WO03/106417,WO04/037171, WO04/058148, WO04/058700, WO04/126699, WO05/018638,WO05/019206, WO05/019205, WO05/018547, WO05/017190, US2005/0176776. Inan embodiment inhibitors of mitotic kinesins include, but are notlimited to inhibitors of KSP, inhibitors of MKLP1, inhibitors of CENP-E,inhibitors of MCAK and inhibitors of Rab6-KIFL.

Examples of “histone deacetylase inhibitors” include, but are notlimited to, SAHA, TSA, oxamflatin, PXD101, MG98 and scriptaid. Furtherreference to other histone deacetylase inhibitors may be found in thefollowing manuscript; Miller, T. A. et al., J. Med. Chem.46(24):5097-5116 (2003).

“Inhibitors of kinases involved in mitotic progression” include, but arenot limited to, inhibitors of aurora kinase, inhibitors of Polo-likekinases (PLK; in particular inhibitors of PLK-1), inhibitors of bub-1and inhibitors of bub-R1. An example of an “aurora kinase inhibitor” isVX-680.

“Antiproliferative agents” includes antisense RNA and DNAoligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001,and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin,doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine,cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed,paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed,nelzarabine, 2′-deoxy-2′-methylidenecytidine,2′-fluoromethylene-2′-deoxycytidine,N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea,N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine,aplidine, ecteinascidin, troxacitabine,4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamicacid, aminopterin, 5-fluorouracil, alanosine,11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-ylacetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N4-palmitoyl-1-B-D-arabino furanosyl cytosine,3-aminopyridine-2-carboxaldehyde thiosemicarbazone and trastuzumab.

Examples of monoclonal antibody targeted therapeutic agents includethose therapeutic agents which have cytotoxic agents or radioisotopesattached to a cancer cell specific or target cell specific monoclonalantibody. Examples include Bexxar.

“HMG-CoA reductase inhibitors” refers to inhibitors of3-hydroxy-3-methylglutaryl-CoA reductase. Examples of HMG-CoA reductaseinhibitors that may be used include but are not limited to lovastatin(MEVACOR®; see U.S. Pat. Nos. 4,231,938, 4,294,926 and 4,319,039),simvastatin (ZOCOR®; see U.S. Pat. Nos. 4,444,784, 4,820,850 and4,916,239), pravastatin (PRAVACHOL®; see U.S. Pat. Nos. 4,346,227,4,537,859, 4,410,629, 5,030,447 and 5,180,589), fluvastatin (LESCOL®;see U.S. Pat. Nos. 5,354,772, 4,911,165, 4,929,437, 5,189,164,5,118,853, 5,290,946 and 5,356,896), atorvastatin (LIPITOR®; see U.S.Pat. Nos. 5,273,995, 4,681,893, 5,489,691 and 5,342,952) andcerivastatin (also known as rivastatin and BAYCHOL®; see U.S. Pat. No.5,177,080). The structural formulas of these and additional HMG-CoAreductase inhibitors that may be used in the instant methods aredescribed at page 87 of M. Yalpani, “Cholesterol Lowering Drugs”,Chemistry & Industry, pp. 85-89 (5 Feb. 1996) and U.S. Pat. Nos.4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitor as usedherein includes all pharmaceutically acceptable lactone and open-acidforms (i.e., where the lactone ring is opened to form the free acid) aswell as salt and ester forms of compounds which have HMG-CoA reductaseinhibitory activity, and therefor the use of such salts, esters,open-acid and lactone forms is included within the scope of thisinvention.

“Prenyl-protein transferase inhibitor” refers to a compound whichinhibits any one or any combination of the prenyl-protein transferaseenzymes, including farnesyl-protein transferase (FPTase),geranylgeranyl-protein transferase type I (GGPTase-I), andgeranylgeranyl-protein transferase type-II (GGPTase-II, also called RabGGPTase).

Examples of prenyl-protein transferase inhibitors can be found in thefollowing publications and patents: WO 96/30343, WO 97/18813, WO97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO95/32987, U.S. Pat. No. 5,420,245, U.S. Pat. No. 5,523,430, U.S. Pat.No. 5,532,359, U.S. Pat. No. 5,510,510, U.S. Pat. No. 5,589,485, U.S.Pat. No. 5,602,098, European Patent Publ. 0 618 221, European PatentPubl. 0 675 112, European Patent Publ. 0 604 181, European Patent Publ.0 696 593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO 95/10515, WO95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO 96/33159, WO96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO97/00252, WO 97/03047, WO 97/03050, WO 97/04785; WO 97/02920, WO97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO98/02436, and U.S. Pat. No. 5,532,359. For an example of the role of aprenyl-protein transferase inhibitor on angiogenesis see European J. ofCancer, Vol. 35, No. 9, pp. 1394-1401 (1999).

“Angiogenesis inhibitors” refers to compounds that inhibit the formationof new blood vessels, regardless of mechanism. Examples of angiogenesisinhibitors include, but are not limited to, tyrosine kinase inhibitors,such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) andFlk-1/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived,or platelet derived growth factors, MMP (matrix metalloprotease)inhibitors, integrin blockers, interferon-α, interleukin-12, pentosanpolysulfate, cyclooxygenase inhibitors, including nonsteroidalanti-inflammatories (NSAIDs) like aspirin and ibuprofen as well asselective cyclooxy-genase-2 inhibitors like celecoxib and rofecoxib(PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch.Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec., Vol. 238, p. 68 (1994);FEBS Letters, Vol. 372, p. 83 (1995); Clin. Orthop. Vol. 313, p. 76(1995); J. Mol. Endocrinol., Vol. 16, p. 107 (1996); Jpn. J. Pharmacol.,Vol. 75, p. 105 (1997); Cancer Res., Vol. 57, p. 1625 (1997); Cell, Vol.93, p. 705 (1998); Intl. J. Mol. Med., Vol. 2, p. 715 (1998); J. Biol.Chem., Vol. 274, p. 9116 (1999)), steroidal anti-inflammatories (such ascorticosteroids, mineralocorticoids, dexamethasone, prednisone,prednisolone, methylpred, betamethasone), carboxyamidotriazole,combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol,thalidomide, angiostatin, troponin-1, angiotensin II antagonists (seeFernandez et al., J. Lab. Clin. Med. 105:141-145 (1985)), and antibodiesto VEGF (see, Nature Biotechnology, Vol. 17, pp. 963-968 (October 1999);Kim et al., Nature, 362, 841-844 (1993); WO 00/44777; and WO 00/61186).

Other therapeutic agents that modulate or inhibit angiogenesis and mayalso be used in combination with the compounds of the instant inventioninclude agents that modulate or inhibit the coagulation and fibrinolysissystems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examplesof such agents that modulate or inhibit the coagulation and fibrinolysispathways include, but are not limited to, heparin (see Thromb. Haemost.80: 10-23 (1998)), low molecular weight heparins and carboxypeptidase Uinhibitors (also known as inhibitors of active thrombin activatablefibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354(2001)). TAFIa inhibitors have been described in U.S. Ser. Nos.60/310,927 (filed Aug. 8, 2001) and 60/349,925 (filed Jan. 18, 2002).

“Agents that interfere with cell cycle checkpoints” refer to compoundsthat inhibit protein kinases that transduce cell cycle checkpointsignals, thereby sensitizing the cancer cell to DNA damaging agents.Such agents include inhibitors of ATR, ATM, the CHK11 and CHK12 kinasesand cdk and cdc kinase inhibitors and are specifically exemplified by7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

“Agents that interfere with receptor tyrosine kinases (RTKs)” refer tocompounds that inhibit RTKs and therefore mechanisms involved inoncogenesis and tumor progression. Such agents include inhibitors ofc-Kit, Eph, PDGF, Flt3 and c-Met. Further agents include inhibitors ofRTKs as described by Bume-Jensen and Hunter, Nature, 411:355-365, 2001.

“Inhibitors of cell proliferation and survival signalling pathway” referto compounds that inhibit signal transduction cascades downstream ofcell surface receptors. Such agents include inhibitors ofserine/threonine kinases (including but not limited to inhibitors of Aktsuch as described in WO 02/083064, WO 02/083139, WO 02/083140, US2004-0116432, WO 02/083138, US 2004-0102360, WO 03/086404, WO 03/086279,WO 03/086394, WO 03/084473, WO 03/086403, WO 2004/041162, WO2004/096131, WO 2004/096129, WO 2004/096135, WO 2004/096130, WO2005/100356, WO 2005/100344, US 2005/029941, US 2005/44294, US2005/43361, 60/734,188, 60/652,737, 60/670,469), inhibitors of Rafkinase (for example BAY-43-9006), inhibitors of MEK (for example CI-1040and PD-098059), inhibitors of mTOR (for example Wyeth CCI-779), andinhibitors of PI3K (for example LY294002).

As described above, the combinations with NSAID's are directed to theuse of NSAID's which are potent COX-2 inhibiting agents. For purposes ofthis specification an NSAID is potent if it possesses an IC₅₀ for theinhibition of COX-2 of 1 μM or less as measured by cell or microsomalassays.

The invention also encompasses combinations with NSAID's which areselective COX-2 inhibitors. For purposes of this specification NSAID'swhich are selective inhibitors of COX-2 are defined as those whichpossess a specificity for inhibiting COX-2 over COX-1 of at least 100fold as measured by the ratio of IC₅₀ for COX-2 over IC₅₀ for COX-1evaluated by cell or microsomal assays. Such compounds include, but arenot limited to those disclosed in U.S. Pat. Nos. 5,474,995, 5,861,419,6,001,843, 6,020,343, 5,409,944, 5,436,265, 5,536,752, 5,550,142,5,604,260, 5,698,584, 5,710,140, WO 94/15932, U.S. Pat. Nos. 5,344,991,5,134,142, 5,380,738, 5,393,790, 5,466,823, 5,633,272 and 5,932,598, allof which are hereby incorporated by reference.

Inhibitors of COX-2 that are particularly useful in the instant methodof treatment are: 3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone;and5-chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine; ora pharmaceutically acceptable salt thereof.

Compounds that have been described as specific inhibitors of COX-2 andare therefore useful in the present invention include, but are notlimited to, the following: parecoxib, BEXTRA® and CELEBREX® or apharmaceutically acceptable salt thereof.

Other examples of angiogenesis inhibitors include, but are not limitedto, endostatin, ukrain, ranpimase, IM862,5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate,acetyldinanaline,5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide,CM101, squalamine, combretastatin, RPI4610, NX31838, sulfatedmannopentaose phosphate,7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalenedisulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone(SU5416).

As used above, “integrin blockers” refers to compounds which selectivelyantagonize, inhibit or counteract binding of a physiological ligand tothe α_(v)β₃ integrin, to compounds which selectively antagonize, inhibitor counteract binding of a physiological ligand to the αvβ5 integrin, tocompounds which antagonize, inhibit or counteract binding of aphysiological ligand to both the α_(v)β₃ integrin and the α_(v)β₅integrin, and to compounds which antagonize, inhibit or counteract theactivity of the particular integrin(s) expressed on capillaryendothelial cells. The term also refers to antagonists of the α_(v)β₆,α_(v)β₈, α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins. The term also refersto antagonists of any combination of α_(v)β₃, α_(v)β₅, α_(v)β₆, α_(v)β₈,α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins.

Some specific examples of tyrosine kinase inhibitors includeN-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline,N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine,BIBX1382,2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one,SH268, genistein, STI571, CEP2563,4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethanesulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI1571A,N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and EMD121974.

Combinations with compounds other than anti-cancer compounds are alsoencompassed in the instant methods. For example, combinations of theinstantly claimed compounds with PPAR-γ (i.e., PPAR-gamma) agonists andPPAR-δ (i.e., PPAR-delta) agonists are useful in the treatment ofcertain malingnancies. PPAR-γ and PPAR-δ are the nuclear peroxisomeproliferator-activated receptors γ and δ. The expression of PPAR-γ onendothelial cells and its involvement in angiogenesis has been reportedin the literature (see J. Cardiovasc. Pharmacol. 1998; 31:909-913; J.Biol. Chem. 1999; 274:9116-9121; Invest. Opthalmol. Vis. Sci. 2000;41:2309-2317). More recently, PPAR-γ agonists have been shown to inhibitthe angiogenic response to VEGF in vitro; both troglitazone androsiglitazone maleate inhibit the development of retinalneovasculatization in mice. (Arch. Ophthamol. 2001; 119:709-717).Examples of PPAR-γ agonists and PPAR-γ/α agonists include, but are notlimited to, thiazolidinediones (such as DRF2725, CS-011, troglitazone,rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate,GW2570, SB219994, AR-H039242, JIT-501, MCC-555, GW2331, GW409544,NN2344, KRP297, NP0110, DRF4158, NN622, G1262570, PNU182716, DRF552926,2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionicacid (disclosed in U.S. Ser. No. 09/782,856), and2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane-2-carboxylicacid (disclosed in U.S. Ser. No. 60/235,708 and 60/244,697).

Another embodiment of the instant invention is the use of the presentlydisclosed compounds in combination with gene therapy for the treatmentof cancer. For an overview of genetic strategies to treating cancer seeHall et al (Am. J. Hum. Genet. 61:785-789, 1997) and Kufe et al (CancerMedicine, 5th Ed, pp 876-889, BC Decker, Hamilton 2000). Gene therapycan be used to deliver any tumor suppressing gene. Examples of suchgenes include, but are not limited to, p 53, which can be delivered viarecombinant virus-mediated gene transfer (see U.S. Pat. No. 6,069,134,for example), a uPA/uPAR antagonist (“Adenovirus-Mediated Delivery of auPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth andDissemination in Mice,” Gene Therapy, August 1998; 5(8):1105-13), andinterferon gamma (J. Immunol. 2000; 164:217-222).

The compounds of the instant invention may also be administered incombination with an inhibitor of inherent multidrug resistance (MDR), inparticular MDR associated with high levels of expression of transporterproteins. Such MDR inhibitors include inhibitors of p-glycoprotein(P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833(valspodar).

A compound of the present invention may be employed in conjunction withanti-emetic agents to treat nausea or emesis, including acute, delayed,late-phase, and anticipatory emesis, which may result from the use of acompound of the present invention, alone or with radiation therapy. Forthe prevention or treatment of emesis, a compound of the presentinvention may be used in conjunction with other anti-emetic agents,especially neurokinin-1 receptor antagonists, 5HT3 receptor antagonists,such as ondansetron, granisetron, tropisetron, and zatisetron, GABABreceptor agonists, such as baclofen, a corticosteroid such as Decadron(dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten orothers such as disclosed in U.S. Pat. Nos. 2,789,118, 2,990,401,3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712, anantidopaminergic, such as the phenothiazines (for exampleprochlorperazine, fluphenazine, thioridazine and mesoridazine),metoclopramide or dronabinol. In another embodiment, conjunctive therapywith an anti-emesis agent selected from a neurokinin-1 receptorantagonist, a 5HT3 receptor antagonist and a corticosteroid is disclosedfor the treatment or prevention of emesis that may result uponadministration of the instant compounds.

Neurokinin-1 receptor antagonists of use in conjunction with thecompounds of the present invention are fully described, for example, inU.S. Pat. Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595,5,459,270, 5,494,926, 5,496,833, 5,637,699, 5,719,147; European PatentPublication Nos. EP 0 360 390, 0 394 989, 0 428 434, 0 429 366, 0 430771, 0 436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0512 902, 0 514 273, 0 514 274, 0 514 275, 0 514 276, 0 515 681, 0 517589, 0 520 555, 0 522 808, 0 528 495, 0 532 456, 0 533 280, 0 536 817, 0545 478, 0 558 156, 0 577 394, 0 585 913, 0 590 152, 0 599 538, 0 610793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674, 0707 006, 0 708 101, 0 709 375, 0 709 376, 0 714 891, 0 723 959, 0 733632 and 0 776 893; PCT International Patent Publication Nos. WO90/05525, 90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151,92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330,93/00331, 93/01159, 93/01165, 93/01169, 93/01170, 93/06099, 93/09116,93/10073, 93/14084, 93/14113, 93/18023, 93/19064, 93/21155, 93/21181,93/23380, 93/24465, 94/00440, 94/01402, 94/02461, 94/02595, 94/03429,94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94/08997, 94/10165,94/10167, 94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767,94/15903, 94/19320, 94/19323, 94/20500, 94/26735, 94/26740, 94/29309,95/02595, 95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549,95/11880, 95/14017, 95/15311, 95/16679, 95/17382, 95/18124, 95/18129,95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418,95/30674, 95/30687, 95/33744, 96/05181, 96/05193, 96/05203, 96/06094,96/07649, 96/10562, 96/16939, 96/18643, 96/20197, 96/21661, 96/29304,96/29317, 96/29326, 96/29328, 96/31214, 96/32385, 96/37489, 97/01553,97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206, 97/19084,97/19942 and 97/21702; and in British Patent Publication Nos. 2 266 529,2 268 931, 2 269 170, 2 269 590, 2 271 774, 2 292 144, 2 293 168, 2 293169, and 2 302 689. The preparation of such compounds is fully describedin the aforementioned patents and publications, which are incorporatedherein by reference.

In an embodiment, the neurokinin-1 receptor antagonist for use inconjunction with the compounds of the present invention is selectedfrom:2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)morpholine,or a pharmaceutically acceptable salt thereof, which is described inU.S. Pat. No. 5,719,147.

A compound of the instant invention may also be administered with anagent useful in the treatment of anemia. Such an anemia treatment agentis, for example, a continuous eythropoiesis receptor activator (such asepoetin alfa).

A compound of the instant invention may also be administered with anagent useful in the treatment of neutropenia. Such a neutropeniatreatment agent is, for example, a hematopoietic growth factor whichregulates the production and function of neutrophils such as a humangranulocyte colony stimulating factor, (G-CSF). Examples of a G-CSFinclude filgrastim.

A compound of the instant invention may also be administered with animmunologic-enhancing drug, such as levamisole, isoprinosine andZadaxin.

A compound of the instant invention may also be useful for treating orpreventing cancer, including bone cancer, in combination withbisphosphonates (understood to include bisphosphonates, diphosphonates,bisphosphonic acids and diphosphonic acids). Examples of bisphosphonatesinclude but are not limited to: etidronate (Didronel), pamidronate(Aredia), alendronate (Fosamax), risedronate (Actonel), zoledronate(Zometa), ibandronate (Boniva), incadronate or cimadronate, clodronate,EB-1053, minodronate, neridronate, piridronate and tiludronate includingany and all pharmaceutically acceptable salts, derivatives, hydrates andmixtures thereof

A compound of the instant invention may also be useful for treating orpreventing breast cancer in combination with aromatase inhibitors.Examples of aromatase inhibitors include but are not limited to:anastrozole, letrozole and exemestane.

A compound of the instant invention may also be useful for treating orpreventing cancer in combination with siRNA therapeutics.

The compounds of the instant invention may also be administered incombination with γ-secretase inhibitors and/or inhibitors of NOTCHsignaling. Such inhibitors include compounds described in WO 01/90084,WO 02/30912, WO 01/70677, WO 03/013506, WO 02/36555, WO 03/093252, WO03/093264, WO 03/093251, WO 03/093253, WO 2004/039800, WO 2004/039370,WO 2005/030731, WO 2005/014553, U.S. Ser. No. 10/957,251, WO2004/089911, WO 02/081435, WO 02/081433, WO 03/018543, WO 2004/031137,WO 20041031139, WO 2004/031138, WO 2004/101538, WO 2004/101539 and WO02/47671 (including LY450139).

A compound of the instant invention may also be useful for treating orpreventing cancer in combination with PARP inhibitors.

A compound of the instant invention may also be useful for treatingcancer in combination with the following therapeutic agents: abarelix(Plenaxis Depot®); aldesleukin (Prokine®); Aldesleukin (Proleukin®);Aleemtuzimabb (Campath®); alitretinoin (Panretin®); alopurinol(Zyloprim®); altretamine (Hexalen®); amifostine (Ethyol®); anastrozole(Arimidex®); arsenic trioxide (Trisenox®); asparaginase (Elspar®);azacitidine (Vidaza®); bevacuzimab (Avastin®); bexarotene capsules(Targretin®); bexarotene gel (Targretin®); bleomycin (Blenoxane®);bortezomib (Velcade®); busulifan intravenous (Busulfex®); busulfan oral(Myleran®); calusterone (Methosarb®); capecitabine (Xeloda®);carboplatin (Paraplatin®); carmustine (BCNU®, BiCNU®); carmustine(Gliadel®); carmustine with Polifeprosan 20 Implant (Gliadel Wafer®);celecoxib (Celebrex®); cetuximab (Erbitux®); chlorambucil (Leukeran®);cisplatin (Platinol®); cladribine (Leustatin®, 2-CdA®); clofarabine(Clolar®); cyclophosphamide (Cytoxan®, Neosar®); cyclophosphamide(Cytoxan Injection®); cyclophosphamide (Cytoxan Tablet®); cytarabine(Cytosar-U®); cytarabine liposomal (DepoCyt®); dacarbazine (DTIC-Dome®);dactinomycin, actinomycin D (Cosmegen®); Darbepoetin alfa (Aranesp®);daunorubicin liposomal (DanuoXome®); daunorubicin, daunomycin(Daunorubicin®); daunorubicin, daunomycin (Cerubidine®); Denileukindiftitox (Ontak®); dexrazoxane (Zinecard®); docetaxel (Taxotere®);doxorubicin (Adriamycin PFS®); doxorubicin (Adriamycin®, Rubex®);doxorubicin (Adriamycin PFS Injection®); doxorubicin liposomal (Doxil®);DROMOSTANOLONE PROPIONATE (DROMOSTANOLONE(D); DROMOSTANOLONE PROPIONATE(MASTERONE INJECTION®); Elliott's B Solution (Elliott's B Solution®);epirubicin (Ellence®); Epoetin alfa (Epogen®); erlotinib (Tarceva®);estramustine (Emcyt®); etoposide phosphate (Etopophos®); etoposide,VP-16 (Vepesid®); exemestane (Aromasin®); Filgrastim (Neupogen®);floxuridine (intraarterial) (FUDR®); fludarabine (Fludara®);fluorouracil, 5-FU (Adrucil®); fulvestrant (Faslodex®); gefitinib(Iressa®); gemcitabine (Gemzar®); gemtuzumab ozogamicin (Mylotarg®);goserelin acetate (Zoladex Implant®); goserelin acetate (Zoladex®);histrelin acetate (Histrelin Implant®); hydroxyurea (Hydrea®);Ibritumoomab Tiuxetan (Zevalin®); idarubicin (Idamycin®); ifosfamide(IFEX®); imatinib mesylate (Gleevec®); interferon alfa 2a (Roferon A®);Interferon alfa-2b (Intron A®); irinotecan (Camptosar®); lenalidomide(Revlimid®); letrozole (Femara®); leucovorin (Wellcovorin®,Leucovorin®); Leuprolide Acetate (Eligard®); levamisole (Ergamisol®);lomustine, CCNU (CeeBU®); meclorethamine, nitrogen mustard (Mustargen®);megestrol acetate (Megace®); melphalan, L-PAM (Alkeran®);mercaptopurine, 6-MP (Purinethol®); mesna (Mesnex®); mesna (MesnexTabs®); methotrexate (Methotrexate®); methoxsalen (Uvadex®); mitomycin C(Mutamycin®); mitotane (Lysodren®); mitoxantrone (Novantrone®);nandrolone phenpropionate (Durabolin-50®); nelarabine (Arranon®);Nofetumnomrab (Verluma®); Oprelvekin (Neurnega®); oxaliplatin(Eloxatin®); paclitaxel (Paxene®); paclitaxel (Taxol®); paclitaxelprotein-bound particles (Abraxane®); palifermin (Kepivance®);pamidronate (Aredia®); pegademase (Adagen (Pegademase Bovine)®);pegaspargase (Oncaspar®); Pegfilgrastim (Neulasta®); pemetrexed disodium(Alimta®); pentostatin (Nipent®); pipobroman (Vercyte®); plicamycin,mithramycin (Mithracin®); porfimer sodium (Photofrin®); procarbazine(Matulane®); quinacrine (Atabrine®); Rasburicase (Elitek®); Rituximab(Rituxan®); sargramostim (Leukine®); Sargramostim (Prokine®); sorafenib(Nexavar®); streptozocin (Zanosar®); sunitinib maleate (Sutent®); talc(Scierosol®); tamoxifen (Nolvadex®); temozolomide (Temodar®);teniposide, VM-26 (Vumon®) testolactone (Teslac®); thioguanine, 6-TG(Thioguanine®); thiotepa (Thioplex®); topotecan (Hycamtin®); toremifene(Fareston®); Tositumomab (Bexxar®); Tositumomab/I-131 tositumomab(Bexxar®); Trastuzumab (Herceptin®); tretinoin, ATRA (Vesanoid®); UracilMustard (Uracil Mustard Capsules®); valrubicin (Vaistar®); vinblastine(Velban®); vincristine (Oncovin®); vinorelbine (Navelbine®); andzoledronate (Zometa®).

Thus, the scope of the instant invention encompasses the use of theinstantly claimed compounds in combination with a second compoundselected from: an estrogen receptor modulator, an androgen receptormodulator, a retinoid receptor modulator, a cytotoxic/cytostatic agent,an antiproliferative agent, a prenyl-protein transferase inhibitor, anHMG-CoA reductase inhibitor, an HIV protease inhibitor, a reversetranscriptase inhibitor, an angiogenesis inhibitor, PPAR-γ agonists,PPAR-δ agonists, an inhibitor of inherent multidrug resistance, ananti-emetic agent, an agent useful in the treatment of anemia, an agentuseful in the treatment of neutropenia, an immunologic-enhancing drug,an inhibitor of cell proliferation and survival signaling, abisphosphonate, an aromatase inhibitor, an siRNA therapeutic,γ-secretase inhibitors, agents that interfere with receptor tyrosinekinases (RTKs), an agent that interferes with a cell cycle checkpointand any of the therapeutic agents listed above.

The term “administration” and variants thereof (e.g., “administering” acompound) in reference to a compound of the invention means introducingthe compound or a prodrug of the compound into the system of the animalin need of treatment. When a compound of the invention or prodrugthereof is provided in combination with one or more other active agents(e.g., a cytotoxic agent, etc.), “administration” and its variants areeach understood to include concurrent and sequential introduction of thecompound or prodrug thereof and other agents.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.

The term “therapeutically effective amount” as used herein means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue, system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician.

The term “treating cancer” or “treatment of cancer” refers toadministration to a mammal afflicted with a cancerous condition andrefers to an effect that alleviates the cancerous condition by killingthe cancerous cells, but also to an effect that results in theinhibition of growth and/or metastasis of the cancer.

In an embodiment, the angiogenesis inhibitor to be used as the secondcompound is selected from a tyrosine kinase inhibitor, an inhibitor ofepidermal-derived growth factor, an inhibitor of fibroblast-derivedgrowth factor, an inhibitor of platelet derived growth factor, an MMP(matrix metalloprotease) inhibitor, an integrin blocker, interferon-α,interleukin-12, pentosan polysulfate, a cyclooxygenase inhibitor,carboxyamidotriazole, combretastatin A-4, squalamine,6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin,troponin-1, or an antibody to VEGF. In an embodiment, the estrogenreceptor modulator is tamoxifen or raloxifene.

Also included in the scope of the claims is a method of treating cancerthat comprises administering a therapeutically effective amount of acompound of the instant invention in combination with radiation therapyand/or in combination with a second compound selected from: an estrogenreceptor modulator, an androgen receptor modulator, a retinoid receptormodulator, a cytotoxiccytostatic agent, an antiproliferative agent, aprenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, anHIV protease inhibitor, a reverse transcriptase inhibitor, anangiogenesis inhibitor, PPAR-γ agonists, PPAR-δ agonists, an inhibitorof inherent multidrug resistance, an anti-emetic agent, an agent usefulin the treatment of anemia, an agent useful in the treatment ofneutropenia, an immunologic-enhancing drug, an inhibitor of cellproliferation and survival signaling, a bisphosphonate, an aromataseinhibitor, an siRNA therapeutic, γ-secretase inhibitors, agents thatinterfere with receptor tyrosine kinases (RTKs), an agent thatinterferes with a cell cycle checkpoint and any of the therapeuticagents listed above.

And yet another embodiment of the invention is a method of treatingcancer that comprises administering a therapeutically effective amountof a compound of the instant invention in combination with paclitaxel ortrastuzumab.

The invention further encompasses a method of treating or preventingcancer that comprises administering a therapeutically effective amountof a compound of the instant invention in combination with a COX-2inhibitor.

The instant invention also includes a pharmaceutical composition usefulfor treating or preventing cancer that comprises a therapeuticallyeffective amount of a compound of the instant invention and a secondcompound selected from: an estrogen receptor modulator, an androgenreceptor modulator, a retinoid receptor modulator, acytotoxic/cytostatic agent, an antiproliferative agent, a prenyl-proteintransferase inhibitor, an HMG-CoA reductase inhibitor, an HIV proteaseinhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor,a PPAR-γ agonist, a PPAR-δ agonist, an inhibitor of cell proliferationand survival signaling, a bisphosphonate, an aromatase inhibitor, ansiRNA therapeutic, γ-secretase inhibitors, agents that interfere withreceptor tyrosine kinases (RTKs), an agent that interferes with a cellcycle checkpoint and any of the therapeutic agents listed above.

All patents, publications and pending patent applications identified arehereby incorporated by reference.

Abbreviations used in the description of the chemistry and in theExamples that follow are: AEBSF (p-aminoethylbenzenesulfonyl fluoride);Boc₂O (di-tert-butyl dicarbonate); BSA (bovine serum albumin); BuLi(n-Butyl lithium); CDCl₃ (chloroform-d); CuI (copper iodide); CuSO₄(copper sulfate); DCE (dichloroethane); DCM (dichloromethane); DEAD(diethyl azodicarboxylate); DMAP (4-aminopyridine); DMF(N,N-dimethylformamide); DMSO (dimethyl sulfoxide); DTT(dithiothreitol); EDTA (ethylene-diamine-tetra-acetic acid); EGTA(ethylene-glycol-tetra-acetic acid); EtOAc (ethyl acetate); EtOH(ethanol); HOAc (acetic acid); HPLC (high-performance liquidchromatography); HRMS (high resolution mass spectrum); LCMS (liquidchromatograph-mass spectrometer); LHMDS (lithiumbis(trimethylsilyl)amide); LRMS (low resolution mass spectrum); MeOH(methanol); MP-B(CN)H₃ (Macroporous cyanoborohydride); NaHCO₃ (sodiumbicarbonate); Na₂SO₄ (sodium sulfate); Na(OAc)₃BH (sodiumtriacetoxyborohydride); NH₄OAc (ammonium acetate); NBS(N-bromosuccinamide); NMP (1-methyl-2-pyrrolidinone); NMR (nuclearmagnetic resonance); PBS (phosphate buffered saline); PCR (polymerasechain reaction); Pd(dppf)([1,1′-bis(diphenylphosphino)ferrocene]palladium); Pd(Ph₃)₄(palladium(0) tetrakis-triphenylphosphine); POCl₃ (phosphorousoxychloride); PS-DIEA (polystyrene diisopropylethylamine); PS—PPh₃(polystyrene-triphenyl phosphine); PTSA (para-toluene sulfonic acid);RaNi (Raney-Nickel); Selectfluor(1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octanebis(tetrafluoroborate); TBAF (tetrabutylammonium fluoride); THF(tetrahydrofuran); TFA (trifluoroacteic acid); and TMSCH₂N₂(trimethylsilyldiazomethane).

The compounds of this invention may be prepared by employing reactionsas shown in the following Reaction Scheme, in addition to other standardmanipulations that are known in the literature or exemplified in theexperimental procedures. The illustrative Reaction Scheme below,therefore, is not limited by the compounds listed or by any particularsubstituents employed for illustrative purposes. Substituent numberingas shown in the Reaction Scheme does not necessarily correlate to thatused in the claims and often, for clarity, a single substituent is shownattached to the compound where multiple substituents are optionallyallowed under the definitions of Formula A hereinabove.

Reactions used to generate the compounds of this invention are preparedby employing reactions as shown in Reaction Scheme I.

Synopsis of Reaction Scheme

As shown in Reaction Scheme I, the aminobenzoic acid can be cyclizedwith ethylchloroacetate to provide benzoxazinone (A-1). This latentelectrophile can be reacted with the sodium anion of methyl acetoacetateto provide acrylate A-2. Upon treatment of sodium methoxide, A-2 can beinternally cyclized and decarboxylated to provide the4-hydroxyquinolinone A-3. Under microwave heating, thepyrazoloquinolinone (A-4) can be formed from hydrazine and catalyticacid. Selective benzyl protection can occur on the pyrazole nitrogen togive N-benzylated-pyrazoloquinolinone (A-5) which can allow foralkylation by a variety of bromides in the presence of cesium carbonateto give the N-alkylated compounds (A-6). The benzyl group can then beremoved by hydrogenation using palladium on carbon to provide A-7. BOCgroup deprotection of alkyl amines with HCl can give the fullyelaborated pyrazoloquinolinone (A-8).

EXAMPLES

Examples provided are intended to assist in a further understanding ofthe invention. Particular materials employed, species and conditions areintended to be further illustrative of the invention and not limitativeof the reasonable scope thereof. The reagents utilized in synthesizingthe compounds depicted are either commercially available or are readilyprepared by one of ordinary skill in the art.

3-amino-5,6,7,8-tetrahydronaphthalene-2-carboxylic acid (1-2)

To a mixture of activated Raney Nickel (200 g of 50% slurry in water onaluminum; 700 mmol, 13 equiv) and 3-amino-2-naphthoic acid (1-1) (10 g,53 mmol, 1 equiv) in 1:1 isopropanol:water (400 mL) at 90° C. was added1% aqueous NaOH solution (200 mL) over 1 h. The reaction mixture wasstirred for 16 h at 90° C. and additional Raney Nickel (50 g, 180 mmol,3.4 equiv) was added and the resulting mixture was heated at 90° C. for24 h. The mixture was filtered and the filtrate was concentrated to 200mL of water and brought to pH=3 with the addition of 1N aqueoushydrochloric acid solution to precipitate the product. The off-whiteprecipitate was filtered, and purified by reverse phase liquidchromatography (H₂O/CH₃CN gradient w/0.1% TFA present) to yield3-amino-5,6,7,8-tetrahydronaphthalene-2-carboxylic acid (1-2) as a whitesolid. LRMS m/z (M+H) 192.2 found, 192.1 required.

2-ethoxy-6,7,8,9-tetrahydro-4H-naphtho[2,3-d][1,3]oxazin-4-one (1-3)

To a solution of 3-amino-5,6,7,8-tetrahydronaphthalene-2-carboxylic acid(1-2) (5.0 g, 26 mmol, 1 equiv) in pyridine (50 mL) at −10° C. undernitrogen was added ethyl chloroformate (10 mL, 11 g, 100 mmol, 4.0equiv) dropwise over 1 h. The resulting solution was stirred from −10°C. to 23° C. over 20 h. The solvent was evaporated under reducedpressure and the solid residue was suspended in water (100 mL) withstirring for 1 h. The precipitate was filtered, washed with water anddried to yield2-ethoxy-6,7,8,9-tetrahydro-4H-naphtho[2,3-d][1,3]oxazin-4-one (1-3) asa yellow solid. LRMS m/z (M+H) 246.4 found, 246.1 required.

methyl(2Z)-2-acetyl-3-{3-[(ethoxycarbonyl)amino]-5,6,7,8-tetrahydronaphthalen-2-yl}-3-hydroxyorop-2-enoate(1-4)

To a dispersion of sodium hydride (95%) (0.80 g, 33 mmol, 2.1 equiv) inanhydrous benzene (100 mL) under nitrogen was added dropwise methylacetoacetate (5.0 mL, 5.4 g, 46 mmol, 2.9 equiv) and the resultingmixture was stirred at 23° C. for 1 h.2-ethoxy-6,7,8,9-tetrahydro-4H-naphtho[2,3-d][1,3]oxazin-4-one (1-3)(3.8 g, 16 mmol, 1 equiv) was added and the reaction mixture was stirredfor 20 h. The excess sodium hydride was quenched with water and theorganic layer was separated, extracted three times with water and thecombined aqueous layers were made acidic with concentrated hydrochloricacid solution. The resulting precipitate was filtered and dried to yieldmethyl(2Z)-2-acetyl-3-{3-[(ethoxycarbonyl)amino]-5,6,7,8-tetrahydronaphthalen-2-yl}-3-hydroxyprop-2-enoate(1-4) as a yellow oil. LRMS m/z (M+H) 362.5 found, 362.2 required.

3-acetyl-4-hydroxy-6,7,8,9-tetrahydrobenzo[r]quinolin-2(1H)-one (1-5)

To a mixture of sodium hydride (95%) (0.70 g, 29 mmol, 2.6 equiv) inbenzene (100 mL) at 23° C. under nitrogen was added methanol (2.5 mL,2.0 g, 62 mmol, 5.4 equiv) dropwise over 10 min. After stirring for anadditional 5 min, methyl(2Z)-2-acetyl-3-{3-[(ethoxycarbonyl)amino]-5,6,7,8-tetrahydronaphthalen-2-yl}-3-hydroxyprop-2-enoate(1-4) (4.1 g, 11 mmol, 1 equiv) was added and the resulting mixture washeated at 72° C. for 20 h. The solvent was evaporated under reducedpressure and the solid residue was suspended in water (50 mL) for 30 minfollowed by 1 N aqueous hydrochloric acid solution (100 mL) for anadditional 30 min with stirring. The solid was filtered, washed withwater (100 mL) and hexanes (100 mL), and dried to yield3-acetyl-4-hydroxy-6,7,8,9-tetrahydrobenzo[g]quinolin-2(1H)-one (1-5) asan off-white solid. LRMS m/z (M+H) 258.4 found, 258.1 required.

3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one(1-6)

A mixture of3-acetyl-4-hydroxy-6,7,8,9-tetrahydrobenzo[g]quinolin-2(1H)-one (1-5)(2.0 g, 7.8 mmol, 1 equiv), hydrazine (0.40 mL, 0.41 g, 13 mmol, 1.6equiv), and catalytic concentrated hydrochloric acid solution (2 drops)in DMA (30 mL) was heated at 140° C. under nitrogen for 16 h. Thesolution was cooled and diluted with water (30 mL). The precipitate wasfiltered, washed with hexanes (3×100 mL), and dried to yield3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one(1-6) as an off-white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 13.51 (s, 1H),10.99 (s, 1H), 7.70 (s, 1H) 7.04 (s, 1H), 2.78 (m, 4H), 2.59 (s, 3H),1.76 (m, 4H). LRMS m/z (M+H) 254.4 found, 254.1 required.

2-benzyl-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one(1-7)

To a mixture of3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one(1-6) (10.2 g, 40.3 mmol, 1 equiv) and potassium carbonate (30.0 g, 217mmol, 5.38 equiv) in DMF (100 mL) under nitrogen was added benzylbromide (35.0 g, 205 mmol, 5.09 equiv) and the resulting mixture wasstirred at 23° C. for 16 h. The reaction mixture was filtered and thefiltered solid was washed with ether (3×200 mL) followed by water (3×250mL) and dried to yield2-benzyl-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one(1-7) as a white solid. LRMS m/z (M+H) 344.2 found, 344.2 required.

tert-butyl 3-bromo-2-fluoropropylcarbamate (1-9)

To a suspension of NBS (20 g, 110 mmol, 3.0 equiv) in dichloromethane(250 mL) was added dropwise triphenylphosphine (28 g, 110 nmol, 3.0equiv) in dichloromethane (100 mL). After addition was complete, thereaction mixture was stirred for 10 min and a solution of tert-butyl2-fluoro-3-hydroxypropylcarbamate (1-8) (7.0 g, 36 mmol, 1 equiv) indichloromethane (100 mL) followed by pyridine (3.5 g, 44 mmol, 1.2equiv) were added. The resulting mixture was stirred at 23° C. undernitrogen for 16 h. The reaction solution was concentrated and purifiedby silica gel chromatography (100% hexanes to 100% ethyl acetate over 1h, analysis by Ninhydrin stain) to yield tert-butyl3-bromo-2-fluoropropylcarbamate (1-9) as a clear oil. ¹H NMR (500 MHz,CDCl₃) δ 4.89-4.68 (m, 2H), 3.55-3.40 (m, 2H), 1.45 (s, 9H).

tert-butyl3-(2-benzyl-3-methyl-4-oxo-2,4,7,8,9,10-hexahydro-5H-benzo[g]pyrazolo[4,3-c]quinolin-5-yl)-2-fluorogropylcarbamate(1-10)

To a mixture of2-benzyl-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one(1-7) (8.0 g, 23 mmol, 1 equiv) and cesium carbonate (37 g, 110 mmol,4.9 equiv) in DMF (100 mL) was added tert-butyl3-bromo-2-fluoropropylcarbamate (1-9) (7.1 g, 27.7 mmol, 1.2 equiv) andthe resulting mixture was stirred at 40° C. for 16 h. The reactionmixture was filtered and the filtrate concentrated and purified bysilica gel chromatography (100% hexanes to 100% ethyl acetate over 1 h)to yield tert-butyl3-(2-benzyl-3-methyl-4-oxo-2,4,7,8,9,10-hexahydro-5H-benzo[g]pyrazolo[4,3-c]quinolin-5-yl)-2-fluoropropylcarbamate(1-10) as a white solid. LRMS m/z (M+H) 519.2 found, 519.3 required.

tert-butyl2-fluoro-3-(3-methyl-4-oxo-2,4,7,8,9,10-hexahydro-5H-benzo[g]pyrazolo[4,3-c]quinolin-5-yl)propylcarbamate(1-11)

A mixture of tert-butyl3-(2-benzyl-3-methyl-4-oxo-2,4,7,8,9,10-hexahydro-5H-benzo[g]pyrazolo[4,3-c]quinolin-5-yl)-2-fluoropropylcarbamate(1-10) (0.10 g, 0.19 mmol, 1 equiv) and 10% palladium on carbon (300 mg)in DMF (10 mL) and methanol (10 mL) was stirred under a hydrogen balloonat 23° C. for 72 h. The resulting suspension was filtered and thefiltrate was concentrated to yield tert-butyl2-fluoro-3-(3-methyl-4-oxo-2,4,7,8,9,10-hexahydro-5H-benzo[g]pyrazolo[4,3-c]quinolin-5-yl)propylcarbamate(1-11). LRMS m/z (M+H) 429.2 found, 429.2 required.

tert-butyl5-{3-[(tert-butoxycarbonyl)amino]-2-fluoropropyl}-3-methyl-4-oxo-4,5,7,8,9,10-hexahydro-2H-benzo[g]pyrazolo[4,3-c]quinoline-2-carboxylate(1-12)

To a solution of tert-butyl2-fluoro-3-(3-methyl-4-oxo-2,4,7,8,9,10-hexahydro-5H-benzo[g]pyrazolo[4,3-]quinolin-5-yl)propylcarbamate(1-11) (0.083 g, 0.19 mmol, 1 equiv) and Boc₂O (0.05 g, 0.23 mmol, 1.2equiv) in DMF (10 mL) was added DMAP (0.005 g, 0.04 mmol, 0.2 equiv) andthe resulting solution was stirred at 23° C. for 3 h. The solution wasconcentrated and the residue purified by silica gel chromatography (100%hexanes to 100% ethyl acetate over 1 h) to yield tert-butyl5-{3-[(tert-butoxycarbonyl)amino]-2-fluoropropyl}-3-methyl-4-oxo-4,5,7,8,9,10-hexahydro-2H-benzo[g]pyrazolo[4,3-c]quinoline-2-carboxylate(1-12) as a white solid. LRMS m/z (M+H) 529.3 found, 529.3 required.

To a solution of a racemic mixture of tert-butyl5-{3-[(tert-butoxycarbonyl)amino]-2-fluoropropyl}-3-methyl-4-oxo-4,5,7,8,9,10-hexahydro-2H-benzo[g]pyrazolo[4,3-c]quinoline-2-carboxylate(1-12, 130 mg, 0.246 mmol) in methanol (10 mL) was added 4 N HClsolution in dioxane (10 mL) and the resulting solution was stirred at23° C. for 16 h. The clear gel precipitate was filtered, and thefiltered solid was washed with ethyl acetate, dissolved in water andconcentrated to yield the hydrochloride salt of a racemic mixture of5-(3-amino-2-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-oneas a white solid (1-12a). ¹H NMR (500 MHz, CD₃OD) δ 7.76 (s, 1H), 7.39(s, 1H), 5.20 (m, 1H), 4.66 (m, 2H), 3.46 (m, 2H), 2.94 (m, 4H), 2.71(s, 3H), 1.87 (m, 4H). LRMS m/z (M+H) 329.2 found, 329.2 required.

tert-butyl5-{3-[(tert-butoxycarbonyl)amino]-2-(R)-fluoropropyl}-3-methyl-4-oxo-4,5,7,8,9,10-hexahydro-2H-benzo[g]pyrazolo[4,3-c]quinoline-2-carboxylate(1-13) and (2-S)-tert-butyl5-{3-[(tert-butoxycarbonyl)amino]-2-(S)-fluoropropyl}-3-methyl-4-oxo-4,5,7,8,9,10-hexahydro-2H-benzo[g]pyrazolo[4,3-c]quinoline-2-carboxylate(1-14)

A racemic mixture of tert-butyl5-{3-[(tert-butoxycarbonyl)amino]-2-fluoropropyl}-3-methyl-4-oxo-4,5,7,8,9,10-hexahydro-2H-benzo[g]pyrazolo[4,3-c]quinoline-2-carboxylate(1-12) was resolved into its constituent enantiomers using a ChiralcelOD (5 cm×50 cm, 201) prep column with an 80% hexane/20% isopropanolgradient at 90 mL/min to give tert-butyl5-{3-[(tert-butoxycarbonyl)amino]-2-(R)-fluoropropyl}-3-methyl-4-oxo-4,5,7,8,9,10-hexahydro-2H-benzo[g]pyrazolo[4,3-]quinoline-2-carboxylate(1-13) and (2-S)-tert-butyl5-{3-[(tert-butoxycarbonyl)amino]-2-(S)-fluoropropyl}-3-methyl-4-oxo-4,5,7,8,9,10-hexahydro-2H-benzo[g]pyrazolo[4,3-]quinoline-2-carboxylate(1-14) as white solids. LRMS m/z (M+H) 529.3 found, 529.3 required.

5-(3-amino-2-(R)-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one(1-15) and5-(3-amino-2-(S)-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one(1-16)

To separate solutions of5-(3-amino-2-(R)-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one(1-13) (0.60 g, 1.1 mmol, 1 equiv) and5-(3-amino-2-(S)-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one(1-14) (0.70 g, 13 mmol, 1 equiv) in methanol (20 mL) was added 4 N HClsolution in dioxane (30 mL) and the resulting solutions were stirred at40° C. for 16 h. The clear gel precipitate was filtered, and thefiltered solid was washed with ethyl acetate, dissolved in water andconcentrated to yield the hydrochloride salts of5-(3-amino-2-(R)-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one(1-15) and5-(3-amino-2-(S)-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one(1-16) as white solids. ¹H NMR (500 MHz, CD₃OD) δ 7.76 (s, 1H), 7.39 (s,1H), 5.20 (m, 1H), 4.66 (m, 2H), 3.46 (m, 2H), 2.94 (m, 4H), 2.71 (s,3H), 1.87 (m, 4H). LRMS m/z (M+H) 329.2 found, 329.2 required.

5-(3-amino-2,2-difluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo[4,3-c]quinolin-4-one(1-17) was prepared using3-[(tert-butoxycarbonyl)amino]-2,2-difluoropropyltrifluoromethanesulfonate as the alkylating agent by the methodsdescribed above.

1-17

5-(3-amino-2,2- difluoropropyl)-3- methyl-2,5,7,8,9,10- hexahydro-4H-benzo[g]pyrazolo[4,3- c]quinolin-4-one LRMS m/zz (M + H) 347.2 found,347.2 required.

Examples 1-9

Examples are provided below to further illustrate different features andadvantages of the present invention. The examples also illustrate usefulmethodology for practicing the invention. These examples do not limitthe claimed invention.

Example 1 Identification of CHK1sv1 Using Real-Time PCR

To facilitate the determination of compound inhibitory properties, it isdesirable to identify variants of the “normal” splicing of exon regionsencoding CHK1. In particular, naturally occurring splicing variationsresulting in the loss of the C-terminal regulatory domain of CHK1 weresought. Deletion of the C-terminus confers greater kinase activity toCHK1 (Chen et al., 2000, Cell 100:681-692; Katsuragi and Sagata, 2004,Mol. Biol. Cell. 15:1680-1689). Exons 2-8 encode the catalytic kinasedomain and exon 9 encodes the linker region. The SQ and C-terminalregulatory domains lie within exons 10-13 (Sanchez et al., 1997,277:1497-1501; Katsuragi and Sagata, 2004, Mol. Biol. Cell.15:1680-1689). Real-time PCR experiments and RT-PCR have been used toidentify and confirm the presence of novel splice variants of human CHK1mRNA. A naturally occurring splice variant which encodes a C-terminaltruncation of the CHK1 inhibitory domain was identified, cloned,expressed and purified for use in a CHK1 kinase assay of utility for thedetermination of compound inhibitory properties.

RT-PCR

The structure of CHK1 mRNA in the region corresponding to exons 8 to 11was determined for RNA extracted from human testis using an RT-PCR basedassay. Total RNA isolated from human testis was obtained from BDBiosciences Clontech (Palo Alto, Calif.). RT-PCR primers were selectedthat were complementary to sequences in exon 8 and exon 11 of thereference exon coding sequences in CHK1 (NM_(—)001274). Based upon thenucleotide sequence of CHK1 mRNA, the CHK1 exon 8 and exon 11 primer set(hereafter CHK1₈₋₁₁ primer set) was expected to amplify a 478 base pairamplicon representing the “reference” CHK1 RNA region. The CHK1₈₋₁₁primer set was expected to amplify a 300 base pair amplicon in atranscript that possessed alternative splicing of exon 9 to exon 11. TheCHK1 exon 8 forward primer has the sequence: 5′ATCAGCAAGAATTACCATTCCAGACATC 3′ (SEQ ID NO 1); and the CHK1 exon 11reverse primer has the sequence: 5′ CATACAACTTTTCTTCCATTGATAGCCC 3′ (SEQID NO 2).

Total RNA from human testis was subjected to a one-step reversetranscription-PCR amplification protocol using the Qiagen, Inc.(Valencia, Calif.), One-Step RT-PCR kit, using the following cyclingconditions:

1) 50° C. for 30 minutes;

2) 95° C. for 15 minutes;

3) 35 cycles of:

-   -   94° C. for 30 seconds;    -   63.5° C. for 40 seconds;    -   72° C. for 50 seconds; then    -   72° C. for 10 minutes.

RT-PCR amplification products (amplicons) were size fractionated on a 2%agarose gel. Selected fragments representing 250 to 350 base pairamplicons were manually extracted from the gel and purified with aQiagen Gel Extraction Kit. The purified amplicon fragments werereamplified with the CHK1₈₋₁₁ primer set, and these amplicons were sizefractionated on an agarose gel. Fragments representing 250 to 350 basepair amplicons were manually extracted from the gel and purified with aQiagen Gel Extraction Kit. The purified amplicon fragments werereamplified with the CHK1₈₋₁₁ primer set once more. Following sizefractionation on an agarose gel and manual extraction of the 250 to 350base pair amplicons, the purified amplicon fragments (Qiagen GelExtraction Kit) were cloned into an Invitrogen pCR2.1 vector using thereagents and instructions provided with the TOPO TA cloning kit(Invitrogen, Carlsbad, Calif.). Clones were then plated in pools of 440colonies per plate, onto 15 plates, for a total of 6600 clones. DNA wasextracted from the pooled 440 colonies from each plate and used astemplate for real-time PCR.

Real-Time PCR/TAQman

To determine the presence of an alternatively spliced isoform to theCHK1 reference protein (NP_(—)001265), a real-time PCR assay was used.

TAQman primers and probes used to detect the CHK1sv1 isoform weredesigned and synthesized as pre-set mixtures (Applied Biosystems, FosterCity, Calif.). The sequences of the TAQman primers and probes used todetect the CHK1 reference form (SEQ ID NOs 3, 4, and 5) and CHK1sv1isoform (SEQ ID NOs 6, 7, and 8) are shown in Table 1. Splice junctionspecific probes were labeled with the 6-FAM fluorophore at the 5′ end(FAM) and a non-fluorescent quencher at the 3′ end (NFQ). Real-time PCRwas performed on human testis cDNA using the TaqMan Universal PCR MasterMix (Applied Biosystems, Foster City, Calif.). The TAQman reactioncontained:

96-well format 384-well format 12.5 μl   5 μl TAQman Universal MasterMix1.25 μl 0.5 μl Primer-probe mix 6.25 μl 2.5 μl H₂O   5 μl   2 μl DNA

TABLE 1 Primers and probes used to detect CHK1 isoforms. Name SEQ ID NOSequence Specificity CHK1 reference forward SEQ ID NO 3GTTACTTGGCACCCCAGGA CHK1 primer reference CHK1 reference reverse SEQ IDNO 4 CATCCAATTTGGTAAAGAATCGTGTCA CHK1 primer reference CHK1 referenceprobe SEQ ID NO 5 FAM-TCCTCACAGAACCCC-NFQ CHK1 reference CHK1sv1 forwardSEQ ID NO 6 GCACATTCAATCCAATTTGGACTTCT CHK1sv1 primer CHK1sv1 reverseprimer SEQ ID NO 7 CATCCAATTTGGTAAAGAATCGTGTCAT CHK1sv1 CHK1sv1 probeSEQ ID NO 8 FAM-CAGTGCTTCTAGAACCC-NFQ CHK1sv1

The TAQman reactions were performed on an ABI Prism 7900HT SequenceDetection System (Applied Biosystems, Foster City, Calif.). Thethermocycling conditions were 50° C. for 2 minutes, 95° C. for 10minutes, and 40 cycles of 95° C. for 15 seconds and 60° C. for 1 minute.Data analysis of the fluorescence emission was performed by the SequenceDetector Software (SDS) (Applied Biosystems, Foster City, Calif.).

Results of the TAQman assay indicated that pooled DNA from 13 out of 15plates appeared to possess clones that represented an alternative exon 9to exon 11 splice junction. DNA from one of these positive pools,representing 440 colonies, was used to transform bacterial host cells.Clones were plated in pools of 55 colonies per plate onto 12 platestotal. The colonies on each of the 12 plates were again pooled and usedfor a TAQman assay. Pooled DNA from 1 out of 12 plates appeared topossess a clone that represented an alternative exon 9 to exon 11 splicejunction. The 55 colonies on this positive plate were individuallyscreened using a TAQman assay, and one clone was identified aspossessing an alternative exon 9 to exon 11 splice junction. Thispositive clone was then sequenced from each end using the CHK1 exon 8forward primer (SEQ ID NO 1) and a different exon 11 reverse primer withthe sequence 5′ TGCATCCAATTTGGTAAAGAATCG 3′ (SEQ ID NO 9).

Sequence analysis of the clone revealed that it matched the expectedsequence for alternative splicing of exon 9 of the CHK1 heteronuclearRNA to exon 11; that is the coding sequence of exon 10 is completelyabsent.

Example 2 Cloning of CHK1sv1

Real-time PCR, RT-PCR, and sequencing data indicate that in addition tothe normal CHK1 reference mRNA sequence, NM_(—)001274, encoding CHK1protein, NP_(—)001265, a novel splice variant form of CHK1 mRNA alsoexist in testis tissue and MOLT-4, and Daudi cell lines.

Clones having a nucleotide sequence comprising the CHK1sv1 splicevariant identified in Example 1 were isolated usingrecombination-mediated plasmid construction in yeast. A set of twoprimer pairs was used to amplify and clone the entire mRNA codingsequences of CHK1sv1. In the case of CHK1sv1, real-time quantitative PCRanalysis indicated that transcripts of this splice variant form werepresent at very low levels. In order to clone CHK1sv1, clones containingcoding sequences of the reference CHK1 (NM_(—)001274) were altered by anadditional recombination step in yeast with 80 base pair linkers thatwere designed to create the desired exon 9 to exon 11 splice junction.

A 5′ “forward” primer and a 3′ “reverse” primer were designed forisolation of full length clones corresponding to CHK1sv1. The 5′“forward” CHK1sv1 primer was designed to have the nucleotide sequence of5′ TTACTGGCTTATCGAAATTAATACGACTCACTATAG GGAGGAGTCATGGCAGTGCCCTTTGT 3′(SEQ ID NO 10) and to have sequences complementary to exon 2 of the CHK1mRNA (NM_(—)001274). The 3′ “reverse” CHK1sv1 primer was designed tohave the nucleotide sequence of 5′ TAGAAGGCACAGTCGAGGCTGATCAGCGGGTTTAAACTCATGCATCCAATTTGGTAAAGAATCG 3′ (SEQ ID NO 11) and to havesequences complementary to exon 11 of the CHK1 mRNA (NM_(—)001274). The40 nucleotides at the 5′ ends of the primer sequences indicated initalics are “tails” that were incorporated into the PCR amplicons andfacilitated subsequent plasmid recombination events in yeast. TheseCHK1sv1 “forward” and “reverse” primers were expected to amplify codingsequences of the reference CHK1 mRNA (NM_(—)001274), which was then usedin a subsequent recombinational cloning step to create CHK1sv1-specificsequence.

RT-PCR

The CHK1sv1 cDNA sequence was cloned using a combination of reversetranscription (RT) and polymerase chain reaction (PCR). Morespecifically, about 25 ng of MOLT-4 cell line mRNA (BD BiosciencesClontech, Palo Alto, Calif.) was reverse transcribed using SuperscriptII (Gibco/Invitrogen, Carlsbad, Calif.) and oligo d(T) primer(RESGEN/Invitrogen, Huntsville, Ala.) according to the Superscript IImanufacturer's instructions. For PCR, 1 μl of the completed RT reactionwas added to 40 μl of water, 5 μl of 10× buffer, 1 μl of dNTPs and 1 μlof enzyme from a Clontech (Palo Alto, Calif.) Advantage 2 PCR kit. PCRwas done in a Gene Amp PCR System 9700 (Applied Biosystems, Foster City,Calif.) using the CHK1sv1 “forward” and “reverse” primers for CHK1sv1(SEQ ID NOs 10,11). After an initial 94° C. denaturation of 1 minute, 35cycles of amplification were performed using a 30 second denaturation at94° C. followed by a 40 second annealing at 63.5° C. and a 50 secondsynthesis at 72° C. The 35 cycles of PCR were followed by a 10 minuteextension at 72° C. The 50 μl reaction was then chilled to 4° C. 10 μlof the resulting reaction product was run on a 1% agarose (Invitrogen,Ultra pure) gel stained with 0.3 μg/ml ethidium bromide (Fisher Biotech,Fair Lawn, N.J.). Nucleic acid bands in the gel were visualized andphotographed on a UV light box to determine if the PCR had yieldedproducts of the expected size, in the case of the CHK1 mRNA, a productof about 1243 base pairs. The remainder of the 50 μl PCR reactions fromMOLT-4 cells was purified using the QIAquik Gel extraction Kit (Qiagen,Valencia, Calif.) following the QIAquik PCR Purification Protocolprovided with the kit. About 50 μl of product obtained from thepurification protocol was concentrated to about 6 μl by drying in aSpeed Vac Plus (SC110A, from Savant, Holbrook, N.Y.) attached to aUniversal Vacuum System 400 (also from Savant) for about 30 minutes onmedium heat.

Cloning and Assembly of CHK1sv1 Full-Length Clones and YeastTransformation

Assembly of the full length CHK1sv1 clone by homologous recombinationcloning in yeast was performed using a cycloheximide-basedcounterselection scheme similar to that described previously by Raymondet al. (2002, Genome Res. 12:190-197).

Assembly of the full-length CHK1sv1 full length clone by homologousrecombination between the 1243 base pair CHK1 amplicon, produced usingthe CHK1sv1 forward and reverse “tailed” primers described earlier, andthe expression vector was performed by simultaneous transformation ofthese pieces into yeast cells. A subsequent recombination step with 80base pair oligonucleotide linkers created the CHK1sv1 exon 9 to exon 11splice junction. All yeast transformation steps described in subsequentparagraphs were performed by electroporation (Raymond et al., 2002Genome Res. 12:190-197).

1 μg of the 1243 base pair CHK1 purified amplicon was cloned directlyinto 100 ng of SrfI-digested pCMR11 by cotransformation of 100 μl ofyeast strain CMY1-5 (Mate URA3Δ, CYH2^(R)). Ura⁺, cycloheximideresistant colonies were selected on Ura-deficient media platescontaining 1 μg/ml cycloheximide (Sigma, St. Louis, Mo.). Standard yeastmedia were used (Sherman, 1991, Methods Enzymol. 194:3-21). Total DNAfrom yeast cell culture containing the CHK1 clone was used to transformE. coli to chloramphenicol (Sigma, St. Louis, Mo.) resistance to preparea large quantity of the recombinant plasmid as described in Hoffman andWinston (1987 Gene 57:267-72). The colonies were picked from the platesinto 2 ml of 2×LB media. These liquid cultures were incubated overnightat 37° C. Plasmid DNA was extracted from these cultures using the Qiagen(Valencia, Calif.) Qiaquik Spin Miniprep kit.

TABLE 2 Composition of pCMR11 plasmid Nucleotide coordinates Functionaldescription of sequence   1-6013 Copy-control ™ E. coli origin ofreplication from pCClFOS (Epicentre Technologies, Madison, WI).6014-7884 Yeast URA3 gene, ARS4 autonomously replicating sequence andCEN6 centromere from pRS316 (Sikorski and Hieter, 1989). 7885-8825Mammalian CMV promoter from InVitrogen (Carlsbad, CA) vectorpcDNA3.1/myc-HIS A.   8826-10,774 Yeast CYH2 gene amplified from strainBY4709 (Brachmann et al. 1998) 10,775-10,782 Engineered SrfI restrictionsite. 10,783-13,556 Mammalian poly-adenylation sites, selectablemarkers, SV40 origin, etc. from pcDNA3.1/myc-HIS A. 13,557-13,596 DNAsequence from InVitrogen vector pENTR11. 13,597-14,561 pCMR11 -specific; chloramphenicol resistance gene from pCClFOS.

To construct the CHK1sv1 clone, 1 μg of 80 base pair linkers shown inTable 3 (SEQ ID NOs 12, 13) that spans the region of the alternativesplicing of exon 9 to exon 11, and 100 ng of BamHI-digested CHK1/pCMR11clone were used to cotransform 100 μl of a cycloheximide sensitive yeaststrain. The overlapping DNA between the linkers and CHK1/pCMR11 clonedictates that most yeast transformants will possess the correctlyassembled construct. Ura⁺, cycloheximide resistant colonies wereselected for subsequent preparation and transformation of E. coli.Plasmid DNA extracted from E. coli was analyzed by restriction digest toconfirm the presence of the alternative splicing of exon 9 to exon 11 inthe CHK1sv1 clone. Eight CHK1sv1 clones were sequenced to confirmidentity, and the clones possessing the appropriate sequences are usedfor protein expression in multiple systems.

TABLE 3 Linkers used to create exon 9 to exon 11 splice junction forCHK1sv1 clone SEQ ID NO Linker Sequence SEQ ID NO 12AATCCAATTTGGACTTCTCTCCAGTAAACAGTGCTTCTAGAACCCCTGGCAGCGGTTGGTCAAAAGAATGACAC GATTCT SEQ ID NO 13AGAATCGTGTCATTCTTTTGACCAACCGCTGCCAGGGGTTCTAGAAGCACTGTTTACTGGAGAGAAGTCCAAAT TGGATTSummary of CHK1sv1 Polynucleotide

The polynucleotide coding sequence of CHK1sv1 mRNA (Seq ID NO 14)contains an open reading frame that encodes a CHK1sv1 protein (SEQ ID NO15) similar to the reference CHK1 protein (NP_(—)001265), but lackingamino acids encoded by a 178 base pair region corresponding to exons 10of the full length coding sequence of reference CHK1 mRNA(NM_(—)001274). The deletion of the 178 base pair region results in ashift of the protein translation reading frame in comparison to thereference CHK1 protein reading frame, creating a carboxy terminalpeptide region that is unique to CHK1sv1 (italicized in Seq ID NO 15).The frameshift also creates a premature termination codon 29 nucleotidesdownstream of the exon 9/exon 11 splice junction. Therefore, the CHK1sv1protein is missing an internal 59 amino acid region corresponding to theamino acid region encoded by exon 10 and is also lacking the amino acidsencoded by the nucleotides downstream of the premature stop codon ascompared to the reference CHK1 (NP_(—)001265). Exon 10 encodes the SQ/TQdomains of CHK1, and exons 11-13 encode the autoinhibitory region(Sanchez et al., 1997, Science 277:1497-1501; Katsuragi and Sagata,2004, Mol. Biol. Cell. 15: 1680-1689). While deletion of theautoinhibitory region confers constitutive activity to the CHK1 kinasedomain, when the SQ/TQ domains are also removed, CHK1 enzymatic activitydecreases (Ng et al., 2004, J. Biol. Chem. 279:8808-8819).

TABLE 4 Nucleotide coding sequence and coded polypeptide for CHK1sv1 SeqID ATGGCAGTGCCCTTTGTGGAAGACTGGGACTTGGTGCAAACCCT NO 14GGGAGAAGGTGCCTATGGAGAAGTTCAACTTGCTGTGAATAGAGTAACTGAAGAAGCAGTCGCAGTGAAGATTGTAGATATGAAGCGTGCCGTAGACTGTCCAGAAAATATTAAGAAAGAGATCTGTATCAATAAAATGCTAAATCATGAAAATGTAGTAAAATTCTATGGTCACAGGAGAGAAGGCAATATCCAATATTTATTTCTGGAGTACTGTAGTGGAGGAGAGCTTTTTGACAGAATAGAGCCAGACATAGGCATGCCTGAACCAGATGCTCAGAGATTCTTCCATCAACTCATGGCAGGGGTGGTTTATCTGCATGGTATTGGAATAACTCACAGGGATATTAAACCAGAAAATCTTCTGTTGGATGAAAGGGATAACCTCAAAATCTCAGACTTTGGCTTGGCAACAGTATTTCGGTATAATAATCGTGAGCGTTTGTTGAACAAGATGTGTGGTACTTTACCATATGTTGCTCCAGAACTTCTGAAGAGAAGAGAATTTCATGCAGAACCAGTTGATGTTTGGTCCTGTGGAATAGTACTTACTGCAATGCTCGCTGGAGAATTGCCATGGGACCAACCCAGTGACAGCTGTCAGGAGTATTCTGACTGGAAAGAAAAAAAAACATACCTCAACCCTTGGAAAAAAATCGATTCTGCTCCTCTAGCTCTGCTGCATAAAATCTTAGTTGAGAATCCATCAGCAAGAATTACCATTCCAGACATCAAAAAAGATAGATGGTACAACAAACCCCTCAAGAAAGGGGCAAAAAGGCCCCGAGTCACTTGAGGTGGTGTGTCAGAGTCTCCCAGTGGATTTTCTAAGCACATTCAATCCAATTTGGACTTCTCTCCAGTAAACAGTGCTTCTAGAACCCCTGGCAGCGGTTGGTCAAAAGAATGA Seq IDMAVPFVEDWDLVQTLGEGAYGEVQLAVNRVTEEAVAVKIVDMKR NO 15AVDCPENIKKEICINKMLNHENVVKFYGHRREGNIQYLFLEYCSGGELFDRIEPDIGMPEPDAQRFFHQLMAGVVYLHGIGITHRDIKPENLLLDERDNLKISDFGLATVFRYNNRERLLNKMCGTLPYVAPELLKRREFHAEPVDVWSCGIVLTAMLAGELPWDQPSDSCQEYSDWKEKKTYLNPWKKIDSAPLALLHKILVENPSARITIPDIKKDRWYNKPLKKGAKRPRVTSGGVSESPSGFSKHIQSNLDFSPVNSAS

Example 3 Expression of CHK1sv1 Protein

The baculovirus gene expression vector system permits protein expressioninsect cells, which are inexpensive and easy to maintain. The proteinsproduced are of similar quality to that in mammalian cells (Miller,1988, Biotechnology 10:457-465; Miller, 1989, Bioessays 11:91-95).Methods of protein expression using the baculovirus expression vectorsin insect cells are known in the art and techniques are discussed inO'Reilly et al., Baculovirus Expression Vectors—A Laboratory Manual, W.H. Freeman and Co., New York, 1992 and Baculovirus Expression VectorSystem Instruction Manual, 6^(th) edition, Pharmingen, San Diego, 1999.

Cloning CHK1sv1 for Insect Cell Expression

To create a CHK1sv1/baculovirus transfer vector construct, theCHK1sv1/pCMR11 clone (see Example 2) was used as template for PCR toamplify the coding sequence of CHK1sv1 (SEQ ID NO 14) using the primerslisted in Table 5 (SEQ ID NOs 16, 17). The primer represented by SEQ IDNO 16 contains an optimal translation initiation sequence immediatelyupstream of the ATG start codon and an upstream EcoRI restriction sitethat become incorporated into the amplicon. The primer represented bySEQ ID NO 17 contains sequence encoding six histidine residuesC-terminal to the CHK1 sv1 coding sequence as well as an EagIrestriction site that become incorporated into the CHK1sv1 amplicon. TheCHK1sv1 amplicon was run on a 1% agarose gel. A selected ampliconfragment of the expected size, in the case of CHK1sv1, a product ofabout 994 base pairs, was manually extracted from the gel and purifiedwith a Qiagen Gel Extraction Kit. The purified amplicon fragment wasdigested with EcoRI and EagI. The EcoRI/EagI-digested amplicon wasligated into the baculovirus transfer vector pVL1393 (Pharmingen, SanDiego, Calif.) which had been digested with EcoRI and EagI anddephosphorylated with alkaline phosphatase. The CHK1sv1/pVL1393construct was then transformed into E. coli strain DH5α. Plasmid DNAextracted from selected from ampicillin resistant colonies was sequencedto confirm identity, and the clones possessing the appropriate sequenceswere used for protein expression in insect cells.

TABLE 5 Primers used to clone CHK1sv1 into baculovirus transfer vectorpVL1393 SEQ ID NO Primer Sequence SEQ ID NO 16CCCGGAATTCACCATGGCAGTGCCCTTTGTGGAAGAC TGG SEQ ID NO 17TGTGTCCGGCCGTCAGTGATGGTGATGGTGATGTTCT TTTGACCAACCGCTGCCInsect Cell Expression of CHK1sv1

The CHK1sv1/pVL1393 construct was co-transfected with linearized AcNPVBaculoGold DNA (Pharmingen, San Diego, Calif.) into SF9 insect cells(Invitrogen, Carlsbad, Calif.). Individual recombinant viruses wereselected by end point dilution. Virus clones were amplified to obtainhigh titer stocks. These virus stocks were used for protein expressiontests in small scale SF9 cultures to verify production of the CHK1 sv1recombinant protein. Transfected SF9 cell lysates were analyzed bypolyacrylamide gel electrophoresis for CHK1sv1 protein expression. TheCHK1sv1 protein was visualized by Commassie staining or by Westernblotting using an anti-CHK1 antibody (G4 antibody; Santa CruzBiotechnology, Inc). Based on expression, an individual virus wasselected for larger scale CHK1sv1 expression. For recombinant proteinexpression on the liter scale, SF9 suspension cultures were grown at 27°C. in Ex-cell 401 serum-free media (JRH Scientific, Lenexa, Kans.) andwere infected with a recombinant virus stock using a multiplicity ofinfection of 0.3 virus per cell. The infected SF9 culture was harvested72 hour following virus transfection, and pelleted by centrifugation.Pellets were stored at −70° C.

Purification of CHK1sv1 Recombinant Protein

Insect cell pellets were lysed with B-PER protein extraction reagent(Pierce, Rockford, Ill.) containing 1 μM microcystin (Sigma, St. Louis,Mo.), 10 μM cypermethrin (EMD Biosciences, San Diego, Calif.), andEDTA-free Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim,Germany) (I tablet/50 ml lysis buffer). All manipulations during proteinpurification were performed at 4° C. Cells were resuspended in the lysisbuffer were stirred for 45 minutes. DNAseI (Roche) was then added to afinal concentration of 200 U/ml and the cell suspension was stirred foran additional 30 minutes. The lysed cell suspension was centrifuged for30 minutes at 30,000 g. The lysis supernatant was decanted andcentrifuged for 30 minutes at 30,000 g. For each 10 ml of clearedsupernatant, 1 ml bed volume of Talon metal affinity resin (Clontech,Palo Alto, Calif.) was added, and the suspension was stirred for 45minutes. The affinity resin/lysate suspension was centrifuged at 5000 gfor 3 minutes and then the supernatant was discarded. The affinity resinwas washed 4× with Buffer A (50 μM Tris, pH 8.0; 250 mM NaCl) using 5×volumes of the resin. The washed resin was resuspended as a 2× slurry inBuffer A and packed into a chromatography column. The resin-packedcolumn was washed with 6× bed volumes of Buffer A. CHK1sv1-His-taggedprotein is eluted from the column using a step-wise gradient ofimidazole in Buffer A. Imidazole concentrations in the 2× bed volumefractions were 5, 10, 20, 30, 40, 50, and 60 mM. Elution fractions wereconcentrated using the Amicon Ultra 15 Centrifugal Filter Device, 30,000Nominal Molecular Weight Limit (Millipore, Billerica, Mass.). Theconcentrated enzyme fractions were diluted 50% in glycerol and stored at−20° C. Fractions were analyzed for the presence of CHK1sv1-His-taggedprotein using polyacrylamide gel electrophoresis followed by Coomrmassiestaining and Western blotting using an anti-CHK1 antibody (G4 antibody;Santa Cruz Biotechnology, Inc). The CHK1sv1 kinase activity of thecolumn fractions was determined using the kinase assay described in thefollowing section.

Example 4 CHK1sv1 Kinase Assay

CHK1sv1 activity was assayed in vitro using a synthetic peptidesubstrate. The phosphopeptide product was quantitated using a HomogenousTime-Resolved Fluorescence (HTRF) assay system (Park et al., 1999, Anal.Biochem. 269:94-104). The reaction mixture contained 40 mM HEPES, pH7.3; 100 mM NaCl; 10 mM MgCl₂; 2 mM dithiothreitol; 0.1% BSA; 0.1 mMATP; 0.5 μM peptide substrate; and 0.1 nM CHK1sv1 enzyme in a finalvolume of 40 μl. The peptide substrate has the amino acid sequence aminoterminus-GGRARTSSFAEPG-carboxy terminus (Synpep, Dublin Calif.) (SEQ IDNO 18) and is biotinylated at the N-terminus. The kinase reaction wasincubated for 30 minutes at 22° C., and then terminated with 60 μlStop/Detection Buffer (40 mM HEPES, pH 7.3; 10 mM EDTA; 0.125% TritonX-100; 1.25% BSA; 250 nM PhycoLink Streptavidin-Allophycocyanin (APC)Conjugate (Prozyme, San Leandro, Calif.); and 0.75 nM GSK3αanti-phosphoserine antibody (Cell Signaling Technologies, Beverly,Mass.; Cat# 9338) labeled with europium-chelate (Perkin Elmer, Boston,Mass.). The reaction was allowed to equilibrate for 2 hours at 22° C.,and relative fluorescent units were read on a Discovery plate reader(Packard Biosciences). Inhibitor compounds are assayed in the reactiondescribed above, to determine compound IC50s. 1 μL of compound dissolvedin DMSO was added to each 40 μL reaction in a half-log dilution seriescovering a range of 1 nM to 100 μM. Relative phospho substrateformation, read as HTRF fluorescence units, is measured over the rangeof compound concentrations and a titration curve generated using a fourparameter sigmoidal fit.

Specific compounds of the instant invention were tested in the assaydescribed above and were found to have IC₅₀ of ≦50 μM against substrate.

Example 5 Inhibition of CHK1 Autophosohorylation in Cells

Inhibitor compounds are assayed for their ability to inhibit CHK1 incells by monitoring CHK1 autophosphorylation in response to DNA damage.H1299 cells (ATCC, Manassas, Va.) are grown in culture medium: RPMI 1640supplemented with 10% fetal bovine serum; 10 mM HEPES; 2 mM L-glutamine;1× non-essential amino acids; and penicillin-streptomycin. Cells fromT-75 flasks are pooled, counted, seeded into 6 well dishes at 200,000cells per well in 2 ml media, and incubated. Serial dilution series ofcompounds in DMSO or DMSO control are added to each well from a 1000×working stock in DMSO and incubated for 2 hr at 37° C. Following the2-hr incubation period, 100 nM camptothecin (EMD Biosciences, San Diego,Calif.) is added from a 200× working stock in PBS to all drug-treatedcells (except one of the high dose wells) and one DMSO control well.After a 4 hour incubation with camptothecin, each well is washed oncewith ice-cold PBS and 300 μL of lysis buffer (50 mM Tris (pH 8.0), 150mM NaCl, 50 mM NaF, 1% NP-40, 0.5% Deoxycholic acid, 0.1% SDS, 0.5 μMNa₃VO₄ and 1× Protease Inhibitor Cocktail Complete—without EDTA (RocheDiagnostics, Mannheim, Germany)) is added to each well. Plates areshaken at 4° C. for 10-15 min and lysates are then transferred to 1.5 mlmicrocentrifuge tubes and frozen at −80° C. Lysates are thawed on iceand cleared by centrifugation at 15,000×g for 20 min and thesupernatants are transferred to clean tubes.

Samples (20 μL) are prepared for gel electrophoresis by addition of 5 μLof 5× sample loading buffer and heat-denaturation for 5 min at 100° C.Samples are electorphoresed in Tris/Glycine SDS-polyacrylamide gels(10%) and proteins are transferred onto PVDF. Blots are then blocked for1 hr in 3% BSA in TBS and probed using an antibody againstphospho-Ser-296 CHK1 (Cell Signaling Technologies—Cat #2346). Boundantibody is visualized using a horseradish peroxidase conjugatedsecondary antibody (goat anti-rabbit Jackson Labs—Cat# 111-035-046) andenhanced chemiluminescence (ECL-plus, Amersham, Piscataway, N.J.). Afterstripping of the first antibody set by incubation in 62.5 mM Tris HCl pH6.7, 2% SDS and 2-mercaptoethanol to 100 μM for 30 min at 55° C., blotsare re-probed for total CHK1, using a CHK1 monoclonal antibody (SantaCruz Biotechnology Inc., Cat# SC-8408). The CHK1 monoclonal is detectedusing a sheep anti-mouse IgG coupled to horseradish peroxidase (AmershamBiosciences, Piscataway, N.J., Cat#NA931) and enhanced chemiluminescence(ECL-plus, Amersham). ECL exposed films are scanned and the intensity ofspecific bands is quantitated with ImageQuant software. Titrations areevaluated for level of phospho-CHK1 (Ser296) signal normalized to totalCHK1 and IC50 values are calculated.

Example 6 Functional Activity of Inhibitors in Checkpoint Escape AssayDNA Damage Arrest

To measure functional activity of CHK1 inhibitors in cells, compoundsare assayed for their ability to abrogate DNA damage induced cell cyclearrest. The assay determines cell phosphonucleolin levels as a measureof the quantity of cells entering M-phase after cell cycle arrestbrought on by the DNA damaging agent camptothecin.

H1299 cells (ATCC, Manassas Va.) are seeded at a density of 5000cells/well in RPMI640 media supplemented with 10% fetal bovine serum.After incubation for 24 hours at 37° C. at 5% CO₂, camptothecin is addedto a final concentration of 200 nM and incubated for 16 hours. An equalvolume of a test compound serial dilution series in growth media plus200 nM camptothecin and 332 nM nocodozole (final concentration: 50ng/ml) is added and incubation at 37° C. is continued for 8 hours-Mediais removed from the wells and 50 μL lysis buffer (20 mM HEPES, pH7.5,150 mM NaCl, 50 mM NaF, 1% Triton X-100, 10% Glycerol, 1× ProteinaseInhibitor Cocktail (Roche Diagnostics, Mannheim Germany), 1 μl/ml DNaseI (Roche Diagnostics), 300 μM Sodium Orthovanadate, 1 μM Microcystin(Sigma, St. Louis, Mo.) added. The plate with lysis buffer is shaken for30 min at 4° C. and frozen (−70° C.) for 20 min. Levels ofphosphonucleolin in the cell lysates is measured using the IGEN Origentechnology (BioVeris Corp., Gaithersburg, Md.).

Detection of Phosphonucleolin in Cell Lysates

4E2 anti-nucleolin antibody (Research Diagnostics Inc., Flanders, N.J.)was biotinylated using Origen Biotin-LC-NHS-Ester (BioVeris Corp.) usingthe protocol described by the manufacturer. Goat anti-mouse antibody(Jackson Immuno Research, West Grove, Pa.) was ruthenylated employing aruthenylation kit (BioVeris Corp.; cat# 110034) according to theprotocol described by the manufacturer. To each well of a 96-well plateis added 25 μL of antibody buffer (phospho buffered saline pH7.2, 1%bovine serum albumin, 0.5% Tween-20) containing 2 μg/ml biotynylated 4E2anti-nucleolin antibody and 0.4 mg/ml streptavidin coated paramagneticDynabeads (BioVeris Corp.) along with 25 μL of cell lysate (above). Theantibodies and lysate are incubated with shaking for 1 hr at roomtemperature. Next, 50 ng of anti-phosphonucleolin TG3 antibody (AppliedNeuroSolutions Inc., Vernon Hills, Ill.) in a volume of 50 μL ofantibody buffer (above) are added to each well of the lysate mix andincubation is continued for 30 min at room temperature. Lastly, 25 μL ofa 240 ng/ml solution of the ruthenylated goat anti-mouse antibody inantibody buffer is added to each well and incubation continued for 3hours at room temperature. The lysate antibody mixtures are read in aBioVeris M-series M8 analyser and EC50s for compound dependent increasesin phosphor-nucleolin are determined.

Example 7 Other Biological Assays

CHK1 Expression and Purification: Recombinant human CHK1 can beexpressed as a fusion protein with glutathione S-transferase at theamino-terminus (GST-CHK1) using standard baculovirus vectors and a(Bac-to-Bac®) insect cell expression system purchased from GIBCO™Invitrogen. Recombinant protein expressed in insect cells can bepurified using glutathione sepharose (Amersham Biotech) using standardprocedures described by the manufacturer.

CHK1 Fluorescense Polarization Assays: CHK1 kinase inhibitors can beidentified using fluorescence polarization to monitor kinase activity.This assay utilizes 10 nM GST-CHK1 and contains 5 mM2-(N-Morpholino)ethanesulfonic acid (MES, pH 6.5), 5 mM magnesiumchloride (MgCl₂), 0.05% Tween®-20, 1 μM adenosine 5′ triphosphate (ATP),2 mM 1,4-Dithio-DL-threitol (DTT), 1 μM peptide substrate(Biotin-ILSRRPSYRKILND-free acid) (SEQ ID NO: 19), 10 nM peptidesubstrate tracer (Fluorescine-GSRRP-pS-YRKI-free acid)(pS=phosphorylated-Serine) (SEQ ID NO: 20), 60 nganti-phospho-CREB(S133) mouse monoclonal IgG purified on Protein Gsepharose from crude mouse ascites purchased from Cell SignallingTechnologies (Beverly, Mass.), 4% dimethyl sulfoxide (DMSO) and 30 μMinhibitor compound. Reactions are incubated at room temperature for 140minutes and terminated by addition of 25 mM EDTA (pH 8.0). Stoppedreactions are incubated for 120 minutes at room temperature andfluorescence polarization values determined using a MolecularDevices/LJL Biosystems Analyst™ AD (Sunnyvale, Calif.) with standardfluorescine settings.

CHK1 SPA Filtration Assay: Assays (25μ.) contain 10 nM GST-CHK1, 10 mMMES, 2 mM DTT, 10 mM MgCl₂, 0.025% Tween®-20, 1 μM peptide substrate(Biotin-ILSRRPSYRKILND-free acid) (SEQ ID NO: 19), 1 μM ATP, 0.1 μCi³³P-γ-ATP (New England Nuclear, NEN) and are reacted for 90 minutes atroom temperature. Reactions are terminated by adding 55 μl of phosphatebuffered saline containing 50 mM EDTA, 6.9 mM ATP, 0.5 mg Scintilationproximity assay (SPA) beads (Amersham Biosciences). Peptide substrate isallowed to bind beads for 10 minutes at room temperature followed byfiltration on a Packard GF/B Unifilter plate and washed with phosphatebuffered saline. Dried plates may are sealed with Topseal™ (NEN) and ³³Pincorporated to peptide substrate using a Packard Topcount®scintillation counter with standard settings for ³³P.

CHK1 FlashPlate® Kinase Assay: Assays (25 μl) contain 8.7 GST-CHK1, 10mM MES, 0.1 mM ethyleneglycol-bis(β-aminoethylether)-N,N,N′,N′-tetracetic acid (EGTA, pH 8.0),2 mM DTT, 0.05% Tween 20, 3 μM peptide substrate(Biotin-ILSRRPSYRKILND-free acid) (SEQ ID NO: 19), 1 μM ATP, 0.4 μCi33P-γ-ATP (NEN) and 4% DMSO. Reactions are incubated for 30 minutes atroom temperature, terminated with 50 μl of 50 mM EDTA. 90 μl of reactionis transferred to streptavidin-coated FlashPlates® (NEN) and incubatedfor 1 hour at room temperature. Plates are washed with phosphatebuffered saline containing 0.01% Tween-20 and 10 mM sodiumpyrophosphate. Plates are dried, sealed with Topseal™ (NEN) and anamount of ³³P incorporated into the peptide substrate measured using aPackard Topcount® NXT™ scintillation counter with standard settings.

CHK1 DELFIA® Kinase Assay: Assays (25 μl) utilize 6.4 mM GST-CHK1containing 25 mM Tris, pH 8.5, 20% glycerol, 50 mM sodium chloride(NaCl), 0.1 Surfact-Amps® 20, 1 μM peptide substrate(Biotin-GLYRSPSMPEN-amide) (SEQ ID NO: 21), 2 mM DTT, 4% DMSO, 12.5 μMATP, 5 mM MgCl₂ and are reacted for 30 minutes at room temperature.Reactions are terminated with 100 μl Stop buffer containing 1% BSA, 10mM Tris, pH 8.0, 150 mM NaCl and 100 mM EDTA. Stopped reactions (100 μl)are transferred to 96 well neutravidin plates (Pierce) to capture thebiotin-peptide substrate during a 30 minute room temperature incubationWells are washed and reacted with 100 μl PerkinElmer Wallac Assay Buffercontaining 21.5 ng/ml anti-phospho-Ser216-Cdc25c rabbit polyclonalantibody from Cell Signalling Technology (Beverly, Mass.) and 292 ng/mleuropium labeled anti-rabbit-IgG for 1 hour at room temperature. Wellsare washed and europium released from the bound antibody by addition ofEnhancement Solution (100 μl) (PerkinElmer Wallac) and detected using aWallac Victor2™ using standard manufacturer settings.

Compounds of the present invention may be tested in the CHK1 FlashPlate®Kinase Assay described above.

WST Assay: HT29, HCT116 (5000 cells/well) or other cells are seeded (75μl) to 96 well clear bottom plates at densities which provide lineargrowth curves for 72 hours. Cells are cultured under sterile conditionsin appropriate media and for HT29 and HCT116 this media is McCoy's 5Acontaining 10% Fetal Bovine Serum (FBS). Following the initial seedingof cells, cells are incubated at 37° C., 5% CO₂ from 17 to 24 hours atwhich time the appropriate DNA damaging agents (camptothicins,5-fluorouracil and etoposide) are added at increasing concentrations toa point which is capable of causing at least 80% cell killing within 48hours. Final volume of all DNA damaging agent and compound additions are25 μl. Assays contain <1% DMSO final. At the same time as DNA damagingagent addition, CHK1 inhibitor compound is added at fixed concentrationsto each DNA damaging agent titration to observe enhancement of cellkilling. Cell viability/cell killing under the conditions describedabove are determined by addition of WST reagent (Roche) according to themanufacturer at 47 hours following DNA damage and CHK1 inhibitorcompound addition and following a 3.5 hour or 2.5 hour incubation at 37°C., 5% CO₂ wherein OD₄₅₀ is measured.

Compounds of the present invention may be tested in the assays describedabove.

Example 8 Other Biological Assays

Other assays that may be utilized to determine biological activity ofthe instant compounds include assays found in the followingpublications: WO 04/080973, WO 02/070494, and WO 03/101444.

Example 9 Blood Pressure Assay

The assay is designed to evaluate acute changes in blood pressure (BP)and heart rate (HR) during i.v. infusion of compounds in conscious rats.Following vehicle, three compound concentrations are each infused for 20minutes in a dose-escalating protocol at a rate of 25 μl/min. Bloodsamples are obtained at the end of each infusion (20 min.) for druglevel determination and mean BP and HR are analyzed during the last 3minutes of each infusion (17-20 min.).

Specific compounds of the instant invention were tested in the assaydescribed above.

1. A compound of the Formula A:

wherein: R¹ is independently selected from H and F, wherein at least oneof R¹ is F; or a pharmaceutically acceptable salt or a stereoisomerthereof.
 2. A compound according to claim 1 which is:5-(3-amino-2-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo [4,3-c]quinolin-4-one;5-(3-amino-2-(R)-fluoropropyl)-3-methyl-2,5 ,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo [4,3-c]quinolin-4-one;5-(3-ammo-2-(S)-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo [4,3-c]quinolin-4-one; and5-(3-amino-2,2-difluoropropyl)-3-methyl-2,5,7,8,9,10hexahydro-4H-benzo[g]pyrazolo [4,3-c]quinolin-4-one; or apharmaceutically acceptable salt or a stereoisomer thereof.
 3. An HClsalt of a compound according to claim 1 which is:5-(3-amino-2-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo [4,3-c]quinolin-4-one;5-(3-amino-2-(R)-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo [4,3-c]quinolin-4-one; and 5-(3-amino-2-(S)-fluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo [4,3-c]quinolin-4-one; or astereoisomer thereof.
 4. A TFA salt of a compound according to claim 1which is: 5-(3-amino-2,2-difluoropropyl)-3-methyl-2,5,7,8,9,10-hexahydro-4H-benzo[g]pyrazolo [4,3-c]quinolin-4-one; or astereoisomer thereof.
 5. A pharmaceutical composition comprising apharmaceutical carrier, and dispersed therein, a therapeuticallyeffective amount of a compound of claim 1.